Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats

C.F. García-Prieto a, H. Pulido-Olmo b,c, G. Ruiz-Hurtado b,c, M. Gil-Ortega a, I. Aranguez b,d, M.A. Rubio e, M. Ruiz-Gayo a, B. Somoza a, M.S. Fernández-Alfonso b,⁎

a b s t r a c t

Genetic obesity models exhibit endothelial dysfunction associated to adenosine monophosphate-activated pro- tein kinase (AMPK) dysregulation. This study aims to assess if mild short-term caloric restriction (CR) restores endothelial AMPK activity leading to an improvement in endothelial function. Twelve-week old Zucker lean and obese (fa/fa) male rats had access to standard chow either ad libitum (AL, n = 8) or 80% of AL (CR, n = 8) for two weeks. Systolic blood pressure was significantly higher in fa/fa AL rats versus lean AL animals, but was normalized by CR. Endothelium-dependent relaxation to acetylcholine (ACh, 10−9 to 10−4 M) was reduced in fa/fa AL compared to control lean AL rats (p b 0.001), and restored by CR. The AMPK activator AICAR (10−5 to 8·10−3 M) elicited a lower relaxation in fa/fa AL rings that was normalized by CR (p b 0.001). Inhibition of PI3K (wortmannin, 10−7 M), Akt (triciribine, 10−5 M), or eNOS (L-NAME, 10−4 M) markedly reduced AICAR- induced relaxation in lean AL, but not in fa/fa AL rats. These inhibitions were restored by CR in Zucker fa/fa rings. These data show that mild short-term CR improves endothelial function and lowers blood pressure in obesity due to the activation of the AMPK–PI3K–Akt–eNOS pathway.

Keywords:
Endothelial dysfunction Endothelial AMPK
Mild short-term caloric restriction Obesity

1. Introduction

AMP-activated protein kinase (AMPK) is a ubiquitously distributed Ser/Thr kinase that acts as a sensor of cell energy status facilitating fat and carbohydrate catabolism in response to physiological stimuli (phys- ical exercise, increase of particular adipokines) and in pathological situ- ations (hyperosmolarity, fuel deprivation, hypoxia-ischemia) [1–3]. Pharmacological activation of AMPK can be achieved by drugs such as AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide), met- formin, thiazolidinediones or statins [4]. At the vascular level, AMPK is expressed in both endothelial (EC) and vascular smooth muscle cells (VSMC). In cultured EC, AMPK activates endothelial nitric oxide synthase (eNOS) phosphorylation, thus increas- ing nitric oxide (NO) release [5,6] through protein kinase B (Akt) [7] or PI3K-Akt [8]. In both conduit [9] and resistance arteries [10,11], activa- tion of endothelial AMPK also induces eNOS phosphorylation. Other- wise, in VSMC, AMPK reduces vascular tone by decreasing the activity of myosin-light chain kinase due to a loss of sensibility of VSMC to intra- cellular Ca++ [12].
A dysregulation in the AMPK signaling pathway in over-nutrition and obesity has been shown to contribute to the development of meta- bolic disorders and endothelial dysfunction [13]. We have recently shown that an 8-week high-fat diet (HFD) reduces endothelial AMPK phosphorylation leading to a down-regulation of the PI3K-Akt-eNOS pathway that correlates with endothelial malfunction [14]. Decreased AMPK activity together with low rates of NO synthesis have been pro- posed to account for the impairment of endothelium-dependent relax- ation in genetic models of obesity, such as the Otsuka Long Evans Tokushima Fatty (OLETF) rats [15] and young obese Zucker rats [16]. AMPK activity is also reduced in aorta from Zucker diabetic fatty (ZDF) rats [17].
Caloric restriction (CR) provides vascular protection in lean models of vascular aging by reducing blood pressure and improving endothelial function [18]. Underlying mechanisms include the enhancement of eNOS expression and activity [19,20] together with the increase of NO production [21–23]. In addition, CR has been shown to reverse endothe- lial dysfunction in a mouse model of diet-induced obesity (DIO) [24], to reduce vascular oxidative stress in OLETF rats [25], and to lower blood pressure in spontaneously hypertensive rats [26]. Several effects of CR are mediated through AMPK activation, such as revascularization in response to ischemia [27], the improvement of vascular compliance and the reduction of blood pressure in hypertensive rats [26], or the re- duction in endothelial lipotoxicity [28,29]. In addition, activation of AMPK by dietary treatments lessens vascular lipotoxicity associated with insulin resistance, obesity and HFD by decreasing the excessive exposure of the endothelium to free fatty-acids [28,30].
It has long been thought that benefits only appear after long-term or lifelong CR [18]. However, in models of vascular aging benefits in endo- thelial function have been observed even within 3 weeks [21,22]. More- over, little is known about the molecular pathway underlying vascular protective effects of CR in obesity. In this context, the hypothesis of this study is that a short-term (2 weeks) and moderate (20%) CR leads to an improvement in endothelial function through an AMPK-eNOS dependent mechanism in obese Zucker fa/fa rats.

2. Materials and methods

2.1. Animals and dietary treatments

Eight-week old male Zucker lean (lean) and Zucker fa/fa (fa/fa) rats were housed under controlled dark-light cycles (12 h/12 h from 8:00 a.m. to 8:00 p.m.) and temperature (22 °C) conditions with stan- dard food and water ad libitum. Animals were housed individually for body weight (BW) and food intake monitoring for four weeks. Then, an- imals were divided into two groups with a similar mean BW, and assigned either to an ad libitum (AL, n = 8) or a calorie restricted diet (CR; 80% of AL n = 8) for two additional weeks. Adjustment of 20% ca- loric restriction was done individually based on a previous food intake values. On the last day, rats were weighed and killed by decapitation. Blood was collected in chilled EDTA-coated polypropylene tubes in the morning (8 a.m.). To minimize alterations in AMPK activity or phos- phorylation, 24h fasting was omitted to avoid lipolysis and any eventual reduction of adipose tissue amount. Biochemical values represent therefore postprandial concentrations. Plasma samples were frozen for biochemical determinations. Adipose tissues were weighed and normalized by tibia length. Thoracic aortas were used both for vascular function and western blot studies. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US Na- tional Institute of Health (NIH publication No. 85-23, revised in 2011) and was approved by the Ethics Committee of Universidad CEU-San Pablo (BFU2011-25303).

2.2. Plasma measurements

Glucose was measured by a spectrophotometric method (Glucose Trinder Method, Roche, Barcelona, Spain). Triglycerides (TG) and non-esterified free fatty acids (NEFA) were determined using the GPO (Biolabo, France) and ACS-ACOD (Wako, Germany) methods, respectively.

2.3. Blood pressure measurements

Systolic (SBP) and diastolic blood pressure (DBP), and heart rate were measured in lean AL, fa/fa AL, lean CR and fa/fa CR rats under anes- thesia (80 mg/kg ketamine hydrochloride and 8 mg/kg xylazine i.p.). A polyethylene cannula (inner diameter 0.58 mm, outside diameter 0.96 mm; Intramedic, USA) was inserted in the right carotid artery and connected to a pressure transducer (Statham, Harvard Apparatus GmbH, Germany). BP and heart rate were recorded in a PowerLab system (ADInstruments, Oxford, UK).

2.4. Vascular reactivity in the thoracic aorta artery

Vascular reactivity was performed in an organ bath setting as previ- ously described [31]. Briefly, aortic rings of 2 mm length were given an optimal resting tension of 1.5 g, which was readjusted every 15 min during a 60 min equilibration period. Before starting the experiment, rings were contracted with 75 mM KCl to assess their contractility. Endothelial integrity was analyzed by addition of acetylcholine (ACh, 10−9–10−4 M) and endothelium-independent response was evaluated in presence of sodium nitroprusside (SNP, 10−10 to 10−5 M) to seg- ments pre-contracted with phenylephrine (Phe, 10−7 M). Segments with more than 60% relaxation to 10−5 M ACh were considered with en- dothelium (+E) and segments with less than 10% relaxation to 10−5 M ACh were considered as endothelium-free (-E).
Protocol 1: Basal vascular AMPK activity was evaluated by measuring the relaxation to ACh (10−9 to 10−4 M) in presence of the AMPK inhib- itor Compound C (10−5 M, 25 min) in aortic rings +E.
Protocol 2: In another set of experiments, both +E and −E segments pre-contracted with Phe (10−7 M) were treated with the AMPK activator AICAR (10−5 to 8·10−3 M). This nucleoside is taken up by the cell and accumulates in the cytoplasm as the monophosphorylated derivative, 5′-aminoimidazole-4-carboxamide-ribonucleoside, an AMP analogue that activates AMPK without disturbing cellular adenine nucleotide ratio [32]. In some experiments, the PI3K inhibitor wortmannin (10−7 M), the Akt inhibitor triciribine (10−5 M), or the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME, 10−4 M), were added 25 min before pre-contraction with Phe (10−7 M).

2.5. Western blot analysis

Aortas from lean and fa/fa rats were isolated and cleaned of blood and perivascular fat. Western blotting was performed as previously de- scribed [33]. Briefly, 30 μg protein samples were separated by SDS-PAGE gels. Primary antibodies anti p-AMPKα (Thr172) and AMPKα (1:1000 final dilution; Cell Signaling Technology, USA), anti PI3Kα-110 (1:800 final dilution; Cell Signaling Technology), and anti p-Akt (Ser473) and Akt (1:1000 final dilution; Cell Signaling Technology) were applied overnight at 4 °C. After washing, appropriate secondary antibodies (anti-rabbit or anti-mouse IgG-peroxidase conjugated) were applied for 1 h at a dilution of 1:5000. Blots were washed, incubated in commer- cial enhanced chemiluminescence reagents (ECL Prime, Amersham Bioscence, UK) and bands were detected by ChemiDoc XRS+ Imaging System (Bio-Rad, USA). To prove equal loadings of samples, blots were re-incubated with β-actin antibody (1:5000 final dilution; Sigma- Aldrich, USA). Blots were quantified using Image Lab 3.0 software (Bio-Rad, USA). Values for p-AMPKα (Thr172) and for p-Akt (Ser473) were normalized with AMPK and Akt, respectively.

2.6. Data analyses

All values are given as mean ± S.E.M. and n denotes the number of animals used in each experiment. Statistical significance was analyzed by using either Student’s t-test or two-way ANOVA followed by Bonferroni post hoc test for comparison between groups. A value of p b 0.05 was considered statistically significant. In vascular reactivity experiments, contractions are expressed as the percentage of contrac- tion produced by 75 mM KCl. Relaxations are expressed as the percent- age ofa previous Phe contraction. The maximum response (Emax values) was calculated by nonlinear regression analyses of each individual con- centration–response curve. Area under the concentration–response curves (AUC) were calculated from the individual concentration–response curve plots. Some results were expressed as differences of area under the concentration–response curves (ΔAUC) in control and experi- mental situations (GraphPad Software, USA).

2.7. Chemicals

ACh was dissolved in saline and Phe and SNP in 0.01% ascorbic acid/ saline (Sigma-Aldrich, Spain). Both L-NAME (Sigma-Aldrich) and AICAR (Toronto Research Chemicals, Canada) were prepared in water. Com- pound C, wortmannin and triciribine were dissolved in dimethyl sulfox- ide and were provided by Sigma-Aldrich.

3. Results

3.1. Animal body weight and metabolic parameters

Both lean AL and fa/fa AL Zucker rats increased BW over the study time (Fig. 1). CR induced a loss of weight in lean rats (6.98 ± 0.7% reduc- tion compared to BW before CR), whereas it halted BW increase in fa/fa rats. The amount of all adipose tissues (AT) was significantly higher in fa/fa AL rats compared to lean AL rats (Table 1). CR induced a reduction in lumbar, mesenteric, and subcutaneous AT, but not in periaortic or subscapular AT amount in both Zucker lean and fa/fa rats. Liver weight was markedly higher in fa/fa AL rats, but was reduced by CR in both lean CR and fa/fa CR groups (Table 1).

3.2. CR improves endothelial-dependent relaxation in Zucker fa/fa rats

As shown in Fig. 2A, relaxation to ACh (10−9 to 10−4 M) was signif- icantly reduced in Zucker fa/fa compared to lean rats. CR improved relaxation to ACh in rings from fa/fa rats up to lean values and did not affect it in Zucker lean animals. Endothelium-independent relaxation elicited by SNP (10−10 to 10−5 M, Fig. 2B) was similar between groups.

3.3. Basal activity of AMPK was increased by CR in Zucker fa/fa rats

Compound C (10−5 M), an inhibitor of AMPK, significantly reduced relaxation to ACh in lean AL rings (Fig. 3A). In contrast, compound C had no effect on ACh-induced relaxation in fa/fa AL rings (Fig. 3B), suggesting a reduction in AMPK activity in arteries from this group. As illustrated in Fig. 3D, CR restored the inhibitory effect of compound C in fa/fa rings but was without effect in lean rings (Fig. 3C). Relaxation to AICAR (10−5 to 8·10−3 M) was significantly lower in fa/fa AL vs lean AL (Fig. 4A), and restored by CR. No difference was observed between lean AL and CR rings.
To rule out a potential nonspecific activity of compound C [34] and AICAR [35], functional results were confirmed by determining levels of p-AMPKα (Thr172). As shown in Fig. 4B, levels of p-AMPKα (Thr172) were significantly lower in arteries from fa/fa AL. CR enhanced p-AMPK immunoreactivity in fa/fa CR but was devoid of effect in lean rings. To address the role of endothelial AMPK, relaxation to AICAR was characterized in segments with (+E) and without (-E) endothelium. Relaxation to AICAR was significantly shifted to the right in −E rings from both lean AL (Fig. 4C) and fa/fa AL rats (Fig. 4D). The effect was, however, less pronounced in fa/fa AL rings, suggesting a reduction in endothelial AMPK activity in arteries from this group. The difference in AUC (ΔAUC) between +E and −E segments, which indirectly re- flects the contribution of endothelial AMPK to relaxation, was lower in fa/fa AL rings (ΔAUClean AL = 0.14 ± 0.03 vs ΔAUCfa/fa AL = 0.06 ± 0.01; Student’s t-test, p b 0.05). CR restored relaxation to AICAR infa/fa CR rings +E with no change in −E rings (ΔAUCfa/faCR = 0.14 ± 0.01; Student’s t-test, p b 0.001) (Fig. 4F). No difference was observed in lean CR rings compared to lean AL (Fig. 4E).

3.4. AMPK activates the endothelial PI3K-Akt-eNOS pathway

Wortmannin (10−7 M), an inhibitor of PI3K, significantly reduced the relaxation to AICAR in lean AL (Fig. 5A), but not in fa/fa AL rings (Fig. 5B). The inhibitory effect of wortmannin was restored in fa/fa CR (Fig. 5D). No effect was observed in lean CR rings (Fig. 5C). Western blot analysis of PI3K-α110 revealed similar protein levels between groups (Fig. 5E). A similar behavior was observed for the Akt inhibitor, triciribine (10−5 M), which impaired relaxation to AICAR specifically in lean AL samples (Fig. 6A and B). The effect of triciribine was also recovered in fa/fa CR (Fig. 6D) with no effect in lean CR rings (Fig. 6C). p-Akt/Akt levels were similar between groups (Fig. 6E). Finally, L-NAME (10−4 M) reduced relaxation to AICAR (10−5 to 8·10−3 M) in all groups (Fig. 7). However, this effect was much less pronounced in fa/fa AL rings (Fig. 7B) and was increased by CR

4. Discussion

The results of this study show that a mild and short-term CR restores impaired endothelial function and normalizes systolic blood pressure in Zucker fa/fa rats. The mechanism underlying these changes appears to be the increase of endothelial AMPK activity and a subsequent activa- tion of the PI3K-Akt-eNOS pathway. These changes are observed even with a moderate reduction in BW, suggesting that a mild CR provides protective effects on endothelial function in obesity beyond weight loss. It has to be noted that young adult (14-week old) Zucker fa/fa rats, as those used in this study, display both a slight endothelial dysfunction and a moderate increased blood pressure values (we only observed an increase in SBP but not in DBP) [36], while endothelial dysfunction and high blood pressure values have been described in older individ- uals, due to an age-related increase in oxidative stress [36–38]. In any case, the decrease of AMPK activity and NO availability found in our model are coincident with previous data suggesting that a low activity of both AMPK and eNOS in mesenteric arteries might contribute to im- pair endothelium-dependent relaxation in young Zucker fa/fa rats [16]. AMPK activity is also reduced in ZDF [17] and in OLETF rats concurrently with a lower NO synthesis in aortic endothelium, thus correlating with endothelial dysfunction [15]. Similar results have been described for DIO models, where impaired endothelial function parallels with a down-regulation of AMPK and decreased NO availability [14,38]. In this scenario, we show in the current study that CR restores endothelial function in aorta of Zucker fa/fa rats through the up-regulation of AMPK activity and NO availability increase.
It is well established that the activation of AMPK induces NO re- lease through eNOS phosphorylation both in cultured EC [5,6] and whole arteries [9–11]. In cultured EC, both Akt [7] and PI3K [8] have been proposed as possible mediators of this action and the en- dothelial AMPK-PI3K-Akt-eNOS pathway has been demonstrated in whole vessels [14]. Moreover, it is known that CR promotes the acti- vation of the AMPK-eNOS pathway that contributes to the revascu- larization in response to ischemia [27], to the improvement of vascular compliance [26], and to the reduction of blood pressure in hypertensive rats [26]. The pathway linking these actions remains, nonetheless, uncharacterized in obese models subjected to CR. In this study, we provide evidence that both PI3K and Akt are involved in the increase of NO availability after AMPK activation, although levels of these proteins are not modified by CR. In contrast to our approach, the type of CR applied in previous studies was quite severe, reaching between 35% for 4 weeks [27] and 40% for 3 weeks [26]. For this reason, our study provides first evidence for the beneficial effect of a mild and short-term CR on endothelial function and blood pressure in obesity. These effects occur through activation of the endothelial AMPK-PI3K-Akt-eNOS pathway, even with a moderate loss of weight.
One alternative mechanism by which CR could stimulate eNOS in aorta is by activating sirtuin-1 (SIRT-1) [21,40]. Although this effect has been attributed to lifelong CR [40], short-term CR (3-weeks) did not modify SIRT-1 expression in aorta [22]. Since in our study CR was milder than in Zanetti’s one (20% vs 26% CR and 2 weeks vs 3 weeks), we assume that SIRT-1 would not be implicated in the improvement of endothelial function in our model.
AMPK dysregulation in obesity has been shown to account for lipotoxicity in the endothelium [41,42]. In fact, fatty-acid ß-oxida- tion is a pivotal source of energy for EC that depends on the activity of the AMPK/acetyl-Coa carboxylase/carnitine palmitoyl transferase 1 [29]. As a consequence, AMPK-dysfunction can lead to the accumu- lation of TG in EC, especially under pathological conditions char- acterized by elevated plasma NEFA, leading to lipotoxicity and endothelial dysfunction (per se or through NEFA release) [43–45]. Otherwise, high levels of NEFA in EC might promote de novo synthe- sis of diacylglycerol, which could i) activate certain isoforms of pro- tein kinase C and stimulate endothelial superoxide production via NADPH oxidase [46], ii) inhibit eNOS activity [47], iii) dysregulate endothelial responsiveness to insulin [30], and iv) increase endothe- lial expression of inflammatory factors by NF-κB activation [48]. In OLETF rats, lower NO synthesis is associated with lipid accumulation and cell apoptosis in the endothelium [15]. In this context, we observe a marked increase of plasma TG levels in Zucker fa/fa rats versus Zucker lean rats, which might contribute to endothelial lipotoxicity. Moreover, CR leads to a significant reduction of plasma TG in fa/fa that might account for a reduction of endothelial lipotoxicity. In fact, the reduction of liver weight caused by CR might be attributable to a reduction in NEFA or TG accumulation in this tissue. Considered together, CR might ameliorate endothelial lipotoxicity by both decreasing plasma TG levels and activating endothelial AMPK [28].
The improvement of endothelial function by CR is one mechanism underlying the reduction in systolic blood pressure. Nonetheless, blood pressure normalization in obese patients has been also related to an adaptation of the sympathetic system resulting in reduction of sympathetic activity [49]. This lowering in blood pressure has been ob- served after 3-week CR and even with moderate reduction of BW [49].
Therefore, we cannot exclude a reduction of sympathetic activity by CR in our model, which might be supported by the marked reduction in heart rate observed in the Zucker fa/fa group. A limitation of the study is the lack of effect of CR observed for many parameters in lean animals. It is well known that CR promotes blood pressure reduction and improvement of endothelial function in lean models [18–23]. This discrepancy might be related to the differences in severity, duration and species of CR models. In fact, most studies use a CR ranging from 26% [22] to 40% [19,23,26], as well as a longer duration of CR, ranging from 3 weeks [22,26] to 12 weeks [19]. Finally, CR has been performed in mice [21,24], and in rats of different strains, i.e. Sprague–Dawley [19], Fischer 344 [22,50], Wistar [23] and spontane- ously hypertensive rats [26].

5. Conclusions

This study provides new insights on the protective effects of CR on endothelial function in obesity. It demonstrates that CR may affect vascular health by both modulating endothelial function through AMPK-PI3K-Akt-eNOS activation and improving risk factors, i.e. blood pressure and plasma TG levels. These effects are observed even with a moderate reduction in body weight. More studies will be necessary to determine if these beneficial changes are maintained with longer pe- riods of CR associated to significant body weight reduction. Our results emphasize the role of endothelial AMPK as a special relevant target in obese patients with endothelial dysfunction.

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