Caloric restriction, physical exercise, and CB1 receptor blockade as an efficient combined strategy for bodyweight control and cardiometabolic status improvement in male rats

1 Vol.:(0123456789) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports Caloric restriction, physical exercise, and CB1 receptor blockade as an efficient combined strategy for bodyweight control and cardiometabolic status improvement in male rats Luisa M. Lopez Trinidad1,3, Rosario Martinez1,3, Garyfallia Kapravelou1, Milagros Galisteo2, Pilar Aranda1, Jesus M. Porres1* & Maria Lopez‑Jurado1 Obesity is critically associated with the development of insulin resistance and related cardiovascular and kidney diseases. Several strategies for weight loss have been developed but most of them exhibit a post‑intervention rebound effect. Here, we aimed to design combined weight‑loss strategies of caloric restriction, physical exercise, and administration of a CB1 receptor blocker to inhibit food intake that also accomplish the objectives of lost‑weight maintenance and improvement of cardiovascular and renal function. Diet‑induced obesity (DIO) was generated in Sprague Dawley rats for 12 weeks to test the effects of single or combined strategies (i.e. caloric restriction, mixed training protocol, and/or administration of appetite suppressant) on caloric intake, body weight, cardiovascular and renal functionality resulting from a weight‑loss intervention period of 3 weeks followed by 6 weeks of weight maintenance. Consumption of a high‑fat diet (HFD) caused a significant increase in body weight (5th week of the experimental period) and led to the development of insulin resistance, cardiovascular, and renal alterations. The different interventions tested, resulted in a significant body weight loss and improved glucose metabolism, aerobic capacity, electrocardiographic parameters, vascular expression of adhesion molecules and inflammatory mediators, and renal functionality, reaching values similar to the control normocaloric group or even improving them. Successful maintenance of lost weight was achieved along a 6‑week maintenance period in addition to adequate health status. In conclusion, the weight‑loss and maintenance intervention strategies tested were efficient at reversing the obesity‑related alterations in body weight, glucose metabolism, aerobic capacity, cardiovascular and renal functionality. The beneficial action was very consistent for caloric restriction and physical exercise, whereas administration of a CB1 receptor blocker complemented the effects of the prior interventions in some parameters like body weight or aerobic capacity, and showed specific actions in renal status, increasing glomerular filtration rate and diuresis. Overall, the novelty of our study relies on the easy implementation of combined strategies for effective weight management that resulted in significant health benefits. Overweight and obesity are defined as abnormal or excessive fat accumulation that increases the risk to develop multiple pathologies. They lead to adverse metabolic effects on blood pressure, cholesterol, triglycerides, and insulin resistance. This compilation of factors is known as metabolic syndrome (MetS)1, which is directly related to cardiovascular disease and the alteration in other vital functions, such as the renal function2. Overweight, OPEN 1Department of Physiology, Institute of Nutrition and Food Technology (INyTA), Centre for Biomedical Research, Centre for Research in Sport and Health (IMUDS), Universidad de Granada, Avda. del Conocimiento S/N. Armilla (18100), Granada, Spain. 2Department of Pharmacology, School of Pharmacy, Biohealth Research Institute, Centre for Biomedical Research, Universidad de Granada, Granada, Spain. 3 These authors contributed equally: Luisa M. Lopez Trinidad and Rosario Martinez. *email: [email protected] 2 Vol:.(1234567890) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ obesity and its related diseases are largely preventable3. In this regard, a comprehensive understanding of MetS may be important for the adequate planning of prevention strategies. Since its components are all reversible, early diagnosis and lifestyle intervention strategies of MetS offer an effective treatment approach, primarily targeting weight management. The control and maintenance of the body weight at a stable level are achieved when there is a balance between food intake and energy expenditure. A complex physiological control system is involved in the maintenance of the energy balance and includes afferent signals from the periphery regarding the state of energy stores, as well as efferent signals affecting energy intake and expenditure4. This regulatory system includes multiple interactions between the gastrointestinal tract, adipose tissue and central nervous system, and is influenced by behavioral, sensorial, autonomic, nutritional and endocrine mechanisms5. The first step in the treatment of obesity is focused on losing the extra-weight and ameliorate the related metabolic alterations. Another important issue for subjects who complete a weight loss program is to avoid the post-intervention rebound effect. The bodyweight regain usually takes place right after the end of weight loss intervention as weight loss programs are just transient6. A multidisciplinary approach is required, including lifestyle modifications7 and, in some cases, the reinforcement with pharmacological treatment. Related to lifestyle interventions there are two core aspects to correct an altered energy balance: diet and physical exercise. Obesity development is often favored by the consumption of unbalanced and hypercaloric diets, so the caloric restriction of a balanced diet providing adequate amounts of nutrients is highly recommended. On the other hand, physical activity plays an essential role in the prevention and treatment of obesity. It contributes to generating a negative energy balance, thus facilitating weight loss and avoiding the rebound effect and subsequent body weight regain8. It is well established that different training protocols induce changes in a variety of molecular mechanisms involved in numerous intracellular pathways related to glucose and lipid metabolism, inflammation, or antioxidant status9. In addition to lifestyle modifications, the prescription of an appropriate pharmacological agent is sometimes recommended. The endocannabinoid system is comprised by cannabinoid receptors 1 and 2 (CB1 and CB2), the two endocannabinoids anandamide and 2-arachidonoylglycerol, and endocannabinoid anabolic and catabolic enzymes10. The endocannabinoid system (ECS) plays a critical role in obesity development in both central and peripheral functions related to energy metabolism11. At the central level, endocannabinoids act as retrograde neuromodulators of synaptic plasticity, and participate in many physiological processes including pain regulation, learning and memory, appetite and food intake, lipogenesis, and cravings12,13. At the peripheral level, endocannabinoids exert a tonic action on lipogenesis and fat accumulation. Therefore, CB1 blockade may result in a food intake-independent decrease in fat mass through lipolysis14. In fact, CB1 blockade has been shown to be effective in ameliorating obesity and related metabolic disorders15. In addition, the endocannabinoid 2-AG prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which per se, instead, stimulate differentiation16. Moreover, antagonism of CB1 receptor reduced human satellite cell proliferation and enhanced the formation of myotubes representing an adjuvant therapy of muscle dystrophies17. In recent years, the discovery of an expanded endocannabinoid system, the endocannabinoidome, which includes several mediators that are biochemically related to the endocannabinoids, and their receptors and metabolic enzymes, has corroborated its complexity and expanded the potential for developing new therapeutic strategies to treat multiple related pathologies including neurological, inflammatory or metabolic alterations10. Given the above mentioned, we hypothesized that our specific combined strategy of lifestyle and pharmacological interventions could provide interesting benefits in the treatment of obesity and its related cardiovascular and renal alterations. Therefore, this study aimed to design these new strategies and test them in an experimental model of DIO. Specifically, we sought to (1) test the effects on body weight, physical performance, glycaemic and lipid profile, and different parameters related to cardiovascular and renal health, of a combined program with caloric restriction, mixed training exercise protocol, and pharmacological treatment with the appetite suppressant AM251, a well-known research tool that effectively blocks CB1 receptors and exhibits a strong food intake inhibition action combined with increased basal metabolic rate and decreased plasma levels of glucose and LDL-cholesterol. (2) study the synergies taking place between the three above-mentioned strategies both on body weight loss and in the maintenance of weight and metabolic, cardiovascular and renal benefits acquired. Figure1. Effects of DIO and weight control interventions on caloric intake, body weight, body weight/ femur length ratio, and hypothalamic gene expression of transcripts involved in the central regulation of food intake and energy balance. Six control experiments were carried out during 21weeks using a standard rat chow diet (SD12, SD15, and SD21) or a high-fat diet to induce obesity (HFD12, HFD15, and HFD21). For intervention trials, rats were divided into 8 groups that were fed the hypercaloric diet to induce obesity for 12weeks, followed by three weeks of intervention with a high protein diet for weight loss (WL15) combined or not with the training protocol (e or s, respectively) and the pharmacological treatment with CB1 receptor blocker AM251 (AM). The intervention period was followed by an additional 6-week weight-maintenance period of dietary treatment with a standard rat chow diet (WM21) combined or not with the training protocol (e or s, respectively) and the pharmacological treatment (AM) to maintain the weight lost during the previous intervention period of three weeks. (A) Experimental design, (B) average daily caloric intake (kcal/day) along the different experimental stages (DIO, weight-loss intervention, and weight-maintenance), (C) average body weight at the end of the different experimental stages, (D) bodyweight/ femur length ratio (g/cm2) at the end of the different experimental stages, (E) hypothalamic gene expression at the end of the weight-loss intervention and weight-maintenance stages. Results are means of eight rats ± SEM depicted by vertical bars. ***P < 0.001 in t-test (12weeks); A,B,C means with different letters are significantly different (ANOVA treatment, P < 0.05; 15 and 21weeks). cfos c-Fos, npy neuropeptide Y, lepr leptin receptor, hcrt orexin A, cnr1 cannabinoid receptor. ▸ 3 Vol.:(0123456789) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ 4 Vol:.(1234567890) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ Figure1. (continued) 5 Vol.:(0123456789) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ Results Combined weight control strategies efficiently achieve weight loss and prevent body weight rebound after treatment. The effects of DIO and different weight-loss and weight-maintenance interventions on daily caloric intake and body weight at the end of the different experimental stages are presented in Fig.1A–C. Throughout the experimental period, and due to the higher caloric content of HFD, a significantly higher caloric intake was exhibited by HFD-treated rats when compared to their normocaloric controls. Likewise, higher body weight was gained by the former animals. During the weight-loss intervention period from week 12–15 of the experimental period, the individual action of caloric restriction, physical exercise, and AM251 administration caused a decrease in caloric intake that led to a concomitant decrease in body weight of treated rats. However, stronger effects were observed when the three interventions were combined. Along the weight-maintenance stage, a stabilization of caloric intake and maintenance of bodyweight among the different intervention groups were achieved below the values shown by the SD and HFD controls. Besides, the training protocol and the administration of the appetite suppressant played a role to avoid the rebound effect on body weight. These beneficial actions on body weight were reflected on a similar trend in body weight/femur length ratio at the different experimental stages, especially when exercise was combined with AM251 administration. (Fig.1C). Such ratio has been considered representative of the body mass index and adequate to establish the degree of obesity and evolution of rats in a long experimental period. The expression of c-fos and Npy increased in animals fed the HFD on weeks 15 and 21 compared to the SD group, and decreased as a result of bodyweight loss. Such decrease was only observable in Npy during the lostweight maintenance stage (Fig.1D). The administration of AM251 showed an inhibitory action on Npy and CB1 receptor. Weight‑loss interventions cause a significant improvement in aerobic capacity and glycemic profile. Overall, DIO caused a significant decrease in aerobic capacity and physical fitness parameters (distance and maximum speed) compared to the SD group at the end of week 21 of experimental period (Fig.2A). The interventions assayed for bodyweight control were efficient at increasing the above-mentioned parameters vs the HFD groups and the combined effects of caloric restriction and physical exercise (WMe) were of especial relevance. Moreover, such effects were potentiated by AM251 administration that resulted in the highest values for all three parameters in the WMeAM group (P < 0.05). Glycemic profile and AUC after an oral glucose overload carried out at the end of every experimental stage are shown in Fig.2B,C. DIO resulted in higher AUC, a higher postprandial blood glucose peak after 30min of administration, and higher values of glycemia during a more prolonged period after glucose overload, when compared to the SD group. These data point to a situation of insulin resistance, which was reversed by the weight-loss/maintenance interventions assayed. Cardiovascular functionality is significantly improved by weight‑loss and remains stable dur‑ ing the maintenance period. The obesity-related increase in body weight led to a hypertrophied heart whereas weight-loss interventions decreased heart weight to similar values as those found in the SD group in all experimental stages (Table1). Similarly, ventricular electrocardiographic parameters were strongly affected in the HFD groups that showed a significant increase in the amplitudes of QRS complex and T wave along the experimental period. The increased amplitudes were reversed upon body weight loss achieved by the different interventions assayed, reaching values that were equal or even inferior to the SD group. About the QTc interval, it was increased by obesity and tended to decrease by caloric restriction and physical exercise except for group WMeAM on week 21. No relevant changes were observed in any of the parameters related to atrial functionality. Plasma activity of CK-MB was increased in animals that consumed the HFD on weeks 15 and 21 and decreased by the different weight-loss interventions in these experimental stages (Table2). Gene expression of transcripts related to vascular adhesion and angiogenesis (Vcam, Sele, Vegfa) were higher in the aorta of HFD vs SD controls after 21weeks of the experimental period (Fig.3A). In contrast, the different weight-loss and maintenance interventions caused a significant decrease in expression levels to values lower than those observed in SD. Such changes were matched by significant improvements in plasma atherogenic index (Fig.3B). Likewise, gene expression of Nos2, related to the inflammatory status, was higher in the aorta of HFD-fed animals and this increment was reversed by weight control interventions that also down-regulated Ptgs2 expression. Obesity‑induced alterations in renal function and antioxidant capacity are reverted by the weight‑loss and maintenance strategies implemented. Creatinine clearance ratio was significantly increased under our experimental conditions by DIO and AM251 administration during the first 2 stages of the experimental period (Table3). Also, the obesity-related increase was associated with incipient albuminuria at 15weeks that became significant at 21weeks (HFD vs SD). Such was not the case with AM251 administration in which no albuminuria was detected. Creatinine clearance ratio also appeared to be affected by age of the animals and increased in SD rats at 21 vs 12 or 15 weeks. In contrast, caloric restriction and physical exercise tended to normalize all the above markers of altered renal functionality although results did not reach statistical significance in all cases. Regarding the urinary volume and parameters related to kidney stone formation, obesity induction led to a considerable decrease in urinary volume and pH as well as increased phosphaturia at weeks 12 and 15, which run in parallel to a decreased calciuria and increased calcemia (Table2). The metabolic status of both minerals tended to be normalized by the weight control strategies implemented. Renal functionality was also affected by 6 Vol:.(1234567890) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ age of the animals, and considerable changes in urinary pH, phosphaturia, calciuria, and renal clearance were evident in the rats fed the SD diet on week 21 compared to weeks 12 and 15. To assess whether obesity-induced changes in renal functionality could be mediated through increased oxidative stress, the kidney activity of antioxidant enzymes and lipid peroxidation were assessed (Table4). Results are complex and numerous interactions are observable among obesity and weight-loss interventions on the former parameters. Consumption of HFD worsened oxidative stress conditions and resulted in higher Mn-SOD and GPX activities in all the stages of the experimental period, whereas Cu/Zn-SOD activity exhibited an HFD-derived increase on week 15 and a reduction on week 21. The effects of weight control interventions differed based on their implementation during the 3weeks of weight-loss or the 6weeks of weight-maintenance. Specifically, on week 21 of experimental period, there was a significant decrease in Mn-SOD activity caused by the combined action of exercise and AM251 administration. Likewise, a marked decrease was also found in GPX activity caused by the administration of AM251, alone or in combination with physical exercise. Discussion The present study was carried out to assess the changes produced by obesity on some cardio-renal functions and to demonstrate the improvement in these functions due to a decrease in bodyweight and maintenance of lost weight. Caloric restriction, physical exercise, and blockade of CB1 receptor strategies were tested in an animal model of DIO. Under our experimental conditions, obesity was successfully established (difference in body weight between normocaloric and HFD-fed animals was equal or greater than 2 standard deviations) from the 5th week of the Figure2. Effects of DIO and weight control interventions on aerobic capacity and glucose metabolism. (A) maximal oxygen consumption, maximum speed, and distance run during an incremental test were measured as markers of long term effects after 21weeks of the experimental period, (B,C) glycemic profile and area under the curve after an oral glucose overload measured at 12, 15 or 21weeks of the experimental period. Results are means of eight rats ± SEM depicted by vertical bars. *P < 0.05 in t-test (12weeks); A,B,C means with different letters are significantly different (ANOVA treatment, P < 0.05; 15 and 21weeks). AUC area under the curve (arbitrary units), SD normocaloric standard diet group, HFD HFD-treated group. Weight-loss interventions (WL) on weeks 13–15: WLs obese rats treated with caloric restriction and no exercise, WLe obese rats treated with caloric restriction in combination with physical exercise, WLsAM obese rats treated with caloric restriction in combination with AM251 administration and no exercise, WLeAM obese rats treated with caloric restriction in combination with physical exercise and AM251 administration. Weight-maintenance interventions (WM) on weeks 16–21: WMs rats treated with diet SD and no exercise, WMe rats treated with diet SD in combination with physical exercise, WMsAM rats treated with diet SD in combination with AM251 administration and no exercise, WMeAM rats treated with diet SD in combination with physical exercise and AM251 administration. 7 Vol.:(0123456789) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ DIO period by consuming an obesogenic diet compared to the normocaloric groups. Other models of DIO have been described18,19. However, it should be highlighted that this dietary combination led to rapid weight gain in the experimental animals. Once obesity was established, the HFD diet continued to be administered along all the stages of the experimental period to clearly show the related alterations. Afterward, during the 12–15-week intervention period, different weight-loss strategies were implemented that resulted in subsequent body weight loss. Such decrease can be a consequence of several interacting factors like the high protein content of the diet due to its thermogenic action and its high levels of satiating soluble dietary fiber, the potential anorexigenic action of physical exercise, the inhibitory action on food intake of AM251 at the hypothalamus and leptin sensitivity which is usually inhibited by the consumption of a high-fat diet20–23. In this context, administration of AM251 has been shown to display dose-dependent decreases in food intake and weight gain, specially at doses of 2–5mg/ kg24. In addition, the inhibitory action of AM251 can be potentiated by co-administration of leptin. Recently, a crosstalk between CB1 and GLP1 receptors has been described that provides new therapies for obesity25. The coadministration of a peripheral CB1 receptor inhibitor with long-acting GLP1R agonists achieves greater reduction in body weight and fat mass than monotherapies by promoting negative energy balance. During 15–21weeks, weight-maintenance strategies were combined revealing that the combination of the three interventions assayed: a certain degree of caloric restriction, physical exercise, and administration of CB1 receptor blocker was the most efficient to successfully maintain body weight and avoid the rebound effect common to other weight-loss treatments. In fact, during this last stage of the experimental period, the mixed training protocol and, especially, AM251 administration, contributed to maintain body weight via down-regulation of hypothalamic transcripts coding for Npy. Administration of AM251 has been reported to produce a significant decrease in the number of neurons expressing orexin A in the hypothalamus26, whereas Orexin-A represses satiety-inducing POMC neurons and contributes to obesity via stimulation of endocannabinoid signaling27. On Table 1. Effects of DIO and weight control interventions on parameters of heart functionality. SD standard rat chow diet, HFD hypercaloric diet for dietary induction of obesity, WLs high protein weight-loss intervention diet with a sedentary lifestyle (weeks 12–15), WLe high protein weight-loss intervention diet with training protocol, WLsAM high protein weight-loss intervention diet with a sedentary lifestyle and pharmacological treatment with AM251, WLeAM high protein weight-loss intervention diet with training protocol and pharmacological treatment with AM251. WMs high protein weight-loss intervention diet with a sedentary lifestyle (weeks 12–15) followed by weight-maintenance stage (weeks 15–21) with SD dietary treatment and sedentary lifestyle, WMe high protein weight-loss intervention diet with training protocol followed by weight-maintenance stage with SD dietary treatment and training protocol, WMsAM high protein weight-loss intervention diet with a sedentary lifestyle and pharmacological treatment with AM251followed by weightmaintenance stage with SD dietary treatment, sedentary lifestyle, and pharmacological treatment with AM251, WMeAM high protein weight-loss intervention diet with training protocol and pharmacological treatment with AM251 followed by weight-maintenance stage with SD dietary treatment, training protocol, and pharmacological treatment with AM251, Bpm bits per minute, QTc corrected QT interval. Results are means of 8 rats. SEM standard error of the mean. *P < 0.05 in t-test (12weeks); a, b, c, means within the same column with different letters are significantly different (ANOVA treatment, P < 0.05; 15 and 21weeks). Bodyweight (g) Heart weight (g) Heart rate (bpm) P wavelength (s) P wave amplitude (mV) PR interval (s) QRS length (s) QRS amplitude (mV) T wave length (s) T wave amplitude (mV) QTc interval (s) 12weeks SD 516.9 1.53 293.7 0.019 0.105 0.027 0.019 1.35 0.023 0.184 0.098 HFD 648.7*** 1.65 290.0 0.020 0.134*0.025 0.020 1.69*0.027 0.208*0.106 SEM 19.7 0.045 7.3 0.002 0.010 0.002 0.003 0.075 0.001 0.035 0.056 15weeks SD 503.3b 1.41a 263.5a 0.029b 0.112a 0.059b 0.021a 1.15a 0.031a 0.220b 0.103a HFD 704.1a 1.96b 242.4a 0.022b 0.066a 0.055b 0.021a 1.76b 0.040c 0.308b 0.128b WLs 566.7c 1.54a 265.3a 0.024ab 0.117a 0.064b 0.021a 0.911a 0.042ab 0.143a 0.121ab WLe 552.5bc 1.50a 260.9a 0.020b 0.130a 0.030a 0.021a 1.15a 0.041bc 0.192a 0.125b WLsAM 544.2bc 1.55a 268.0a 0.025ab 0.119a 0.065b 0.020a 0.885a 0.064d 0.188a 0.171c WLeAM 511.5bc 1.45a 254.4a 0.019b 0.106a 0.059b 0.020a 1.12a 0.041bc 0.161a 0.123b SEM 21.2 0.09 7.8 0.002 0.041 0.003 0.001 0.105 0.002 0.015 0.006 21weeks SD 631.7a 1.69a 267.9a 0.021ab 0.089a 0.061ab 0.023ab 1.64b 0.042a 0.176a 0.134ab HFD 803.8b 2.02b 274.5a 0.023b 0.129b 0.063ab 0.021a 1.97c 0.049a 0.291b 0.151b WMs 634.2a 1.73a 263.7a 0.020ab 0.103ab 0.061ab 0.021a 1.09a 0.039a 0.203a 0.124a WMe 574.4a 1.64a 255.9a 0.020ab 0.127a 0.067b 0.027b 1.14a 0.039a 0.206a 0.135ab WMsAM 546.4a 1.72a 257.1a 0.022b 0.076a 0.057a 0.022ab 1.32a 0.045a 0.186a 0.134ab WMeAM 590.9a 1.64a 273.9a 0.017c 0.095b 0.054a 0.021a 1.28a 0.049a 0.211a 0.152b SEM 41.8 0.093 6.5 0.001 0.010 0.002 0.002 0.102 0.004 0.011 0.008 8 Vol:.(1234567890) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ the other hand, Di Marzo etal.28 reported that defective leptin signalling is associated with elevated hypothalamic levels of endocannabinoids in obese db/db and ob/ob mice and Zucker rats, whereas acute leptin treatment of normal rats and ob/ob mice reduces the endocannabinoids anandamide and 2-arachidonoyl glycerol in the hypothalamus. DIO after consumption of HFD during 12weeks resulted in the instauration of insulin resistance, as it is shown by the higher AUC after an oral glucose overload compared to SD groups Nevertheless, this pathological situation was reversed by weight loss interventions and AUC returned to values similar to those of SD animals. Other strategies including legume-derived functional ingredients and/or physical exercise29 have also confirmed this beneficial action. Lost weight maintenance further improved the glycemic profile of the animals after the combined action of physical exercise and AM251 administration, enhancing metabolic activities that promote insulin sensitivity. The CB1 receptor is highly expressed in central and peripheral nervous system, as well as various peripheral tissues30,31. Evidence suggests that endocannabinoids are directly involved in the control of food intake and energy utilization by targeting these central and peripheral sites, including skeletal muscle, liver, and adipose tissue32. The effects of AM251 on glucose metabolism can be explained in a similar way to other CB1 receptor inverse agonists33 via metabolic “peripheral” action in adipose tissue in addition to its known “central” effect on food intake, thus providing an effective strategy for improving insulin sensitivity. The observed beneficial action of AM251 is also in agreement with the results of Esposito etal.34, who concluded that modulation of CB1 receptor regulated uptake at the level of the PI3K signaling system in skeletal muscle cells. Therefore, interfering with CB1 signaling could ameliorate glucoregulatory functions in peripheral tissues. In a similar way, Crespillo etal.35 suggested that blockade of CB1 receptor could play an important role in the restoration of compromised metabolic status caused by high fat intake and improve cardiometabolic risk factors. Furthermore, in a recent Table 2. Effects of DIO and weight control interventions on plasma parameters of heart and kidney functionality. SD standard rat chow diet, HFD hypercaloric diet for dietary induction of obesity, WLs high protein weight-loss intervention diet with a sedentary lifestyle (weeks 12–15), WLe high protein weight-loss intervention diet with training protocol, WLsAM high protein weight-loss intervention diet with a sedentary lifestyle and pharmacological treatment with AM251, WLeAM high protein weight-loss intervention diet with training protocol and pharmacological treatment with AM251. WMs high protein weight-loss intervention diet with a sedentary lifestyle (weeks 12–15) followed by weight-maintenance stage (weeks 15–21) with SD dietary treatment and sedentary lifestyle, WMe high protein weight-loss intervention diet with training protocol followed by weight-maintenance stage with SD dietary treatment and training protocol, WMsAM high protein weight-loss intervention diet with a sedentary lifestyle and pharmacological treatment with AM251 followed by weight-maintenance stage with SD dietary treatment, sedentary lifestyle, and pharmacological treatment with AM251, WMeAM high protein weight-loss intervention diet with training protocol and pharmacological treatment with AM251 followed by weight-maintenance stage with SD dietary treatment, training protocol, and pharmacological treatment with AM251. CK-MB creatine kinase MB. Results are means of 8 rats. SEM, standard error of the mean. *P < 0.05, ***P < 0.001 in t-test (12weeks); a, b, c, means within the same column with different letters are significantly different (ANOVA treatment, P < 0.05; 15 and 21weeks). CK-MB (U/dL) ACE (U/dL) Total Proteins (g/dL) Albumin (g/dL) Urea (mg/dL) Uric Acid (mg/ dL) Creatinine (mg/ dL) Phosphorus (mg/dL) Calcium (mg/ dL) 12weeks SD 395.6 69.1 5.87 2.66 28.0 0.95 0.57 6.08 6.41 HFD 292.4 81.8 6.17* 2.92 32.7* 0.69 0.11 5.95 9.80*** SEM 0.16 0.67 0.14 0.15 0.16 0.16 0.08 0.41 0.67 15weeks SD 358.9a 73.4a 6.22c 3.33b 28.2a 0.94ab 0.71bc 6.13b 6.33a HFD 987.0b 73.6a 6.51c 3.08ab 31.0a 0.90ab 0.15a 5.40ab 10.1b WLs 335.6a 57.5b 5.57a 2.88ab 28.0a 0.72a 0.49abc 4.93a 6.59a WLe 362.1a 77.3a 5.85ab 2.86ab 29.7a 0.92ab 0.81c 5.45ab 6.73a WLsAM 213.6a 72.3a 5.34a 2.61a 34.0a 1.02ab 0.40ab 5.41ab 10.8b WLeAM 196.1a 79.8a 5.42a 2.73ab 26.2a 1.19b 0.40ab 6.16b 11.0b SEM 154.0 9.42 0.18 0.21 3.10 0.15 0.13 0.30 0.86 21weeks SD 166.7a 65.0a 6.39a 2.92ab 32.3b 0.78a 0.054a 6.28a 8.68a HFD 571.7b 70.8b 6.07a 2.94ab 24.3ab 1.07a 0.046a 5.93a 9.51ab WMs 206.4a 85.7bc 6.24a 3.09ab 24.0a 1.29ab 0.47a 5.59a 7.86a WMe 207.6a 96.7d 6.09a 3.37b 26.6ab 0.86a 0.55a 5.99a 7.79a WMsAM 283.0a 72.5b 6.00a 2.60a 25.6ab 1.29ab 0.54a 5.85a 11.1b WMeAM 254.0a 75.4b 5.74a 3.09ab 40.6c 1.24ab 0.55a 5.63a 10.1ab SEM 307.4 11.8 0.23 0.22 2.70 0.24 0.67 0.57 0.99 9 Vol.:(0123456789) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ study, Eid etal.36 have tested the effects of two neutral CB1 receptor antagonists (central and peripheral) with improved safety profiles on a preclinical model of insulin resistance. Both compounds alleviated insulin resistance peripherally, and exerted similar effects on rats with metabolic syndrome. They also displayed anti-dyslipidemic, anti-hyperuricemic and anti-inflammatory effects. Consumption of a diet rich in saturated fats has been associated with changes in heart weight running in parallel to the development of obesity and insulin resistance and may cause ventricular modifications, increasing left ventricle mass that will, in turn, lead to diastolic and systolic alterations and modified left ventricle ejection ratio37. Those changes are reflected in electrocardiographic modifications like the increased amplitude of QRS complex or QTc interval related to cardiac pathology and ventricular arrhythmia38. Here, HFD-induced alterations in cardiac functionality appeared to be mostly related to the degree of cardiac hypertrophy and took place mainly at the ventricular level, resulting in the higher amplitude of QRS complex and T-wave as well as lengthening of the corrected QT interval. Pathological alteration of these parameters indicates disturbances in the electrical activity of the heart and, consequently, on the efficiency of pumping blood. Here, such changes began to show a trend at 12weeks and were significant at 15weeks. We have not observed any changes in ECG parameters related to atrial depolarization and conduction (P wavelength, P wave amplitude, or PR interval), reinforcing the idea that obesity affected mainly ventricular function. Modifications in ECG (QRS amplitude and T wave amplitude) were corrected after the weight loss interventions, although no additional effect to that of caloric restriction was achieved by exercise, CB1 receptor blockade, or the combination of both interventions. Furthermore, no significant effect of exercise was found on QTc interval in a similar way to what has been reported in the obese Zucker rat model39. The improvement in parameters of cardiovascular health achieved by Figure3. Effects of DIO and weight control interventions on vascular damage measured in the aorta and plasma. (A) gene expression of transcripts coding for vascular adhesion molecules and inflammation markers at the end of the 21-week experimental period, (B) plasma atherogenic index at the end of the different experimental stages. Results are means of eight rats ± SEM depicted by vertical bars. *P < 0.05 in t-test (12weeks); A,B,C means with different letters are significantly different (ANOVA treatment, P < 0.05; 15 and 21weeks). Vcam vascular cell adhesion molecule 1, Vegfa Vascular endothelial growth factor A, Sele Selectin E, Nos2 Nitric oxide synthase 2, Ptgs2 Prostaglandin-endoperoxide synthase 2. Atherogenic index, (totalcholesterol (mg/dL)/HDL-cholesterol (mg/dL). SD normocaloric standard diet group, HFD HFD-treated group. Weight loss interventions (WL) on weeks 13–15: WLs obese rats treated with caloric restriction and no exercise, WLe obese rats treated with caloric restriction in combination with physical exercise, WLsAM obese rats treated with caloric restriction in combination with AM251 administration and no exercise, WLeAM obese rats treated with caloric restriction in combination with physical exercise and AM251 administration. Weight-maintenance interventions (WM) on weeks 16–21: WMs rats treated with diet SD and no exercise, WMe rats treated with diet SD in combination with physical exercise, WMsAM rats treated with diet SD in combination with AM251 administration and no exercise, WMeAM rats treated with diet SD in combination with physical exercise and AM251 administration. 16 Vol:.(1234567890) Scientific Reports | (2021) 11:4286 | https://doi.org/10.1038/s41598-021-83709-9 www.nature.com/scientificreports/ 60. Coll-Risco, I. et al. Effects of interval aerobic training combined with strength exercise on body composition, glycaemic and lipid profile and aerobic capacity of obese rats. J. Sports. Sci. 34(15), 1452–1460 (2016). 61. Martínez, R. et al. Effects of a combined intervention with a lentil protein hydrolysate and a mixed training protocol on the lipid metabolism and hepatic markers of NAFLD in Zucker rats. Food. Funct. 9(2), 830–850 (2018). 62. Wisløff, U., Helgerud, J., Kemi, O. J. & Ellingsen, Ø. Intensity-controlled treadmill running in rats: VO2 max and cardiac hypertrophy. Am. J. Physiol. Heart. Circ. Physiol. 280(3), H1301–H1310 (2001). 63. Clemente, V. J. C., Martín, S., Porres, J., Fuentes, S. & Ramírez, P. A. Efecto de la suplementación de vitamina en el rendimiento de una prueba incremental de consumo máximo de oxígeno en ratas Wistar. Arch. Med. Dep. XXVIII, 168–173 (2011). 64. Prieto, P. G., Cancelas, J., Villanueva-Peñacarrillo, M. L., Valverde, I. & Malaisse, W. J. Plasma D-glucose, D-fructose and insulin responses after oral administration of D-glucose, D-fructose and sucrose to normal rats. J. Am. Coll. Nutr. 23, 414–419 (2004). 65. Pruessner, J. C., Kirschbaum, C., Meinlschmid, G. & Hellhammer, D. H. Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change. Psychoneuroendocrinology. 28(7), 916–931 (2003). 66. Porres, J. M. et al. The combined treatment with lentil protein hydrolysate and a mixed training protocol is an efficient lifestyle intervention to manage cardiovascular and renal alterations in obese Zucker rats. Eur. J. Nutr. 59, 3473–3490 (2020). 67. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))-DDCt Method. Methods 25, 402–408 (2001). 68. Cohen, G., Kim, M. & Ogwu, V. A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures. J. Neurosci. Methods. 67(1), 53–56 (1996). 69. Lawrence, R. A., Sunde, R. A., Schwartz, G. L. & Hoekstra, W. G. Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake. Exp. Eye. Res. 18(6), 563–569 (1974). 70. Ukeda, H., Maeda, S., Ishii, T. & Sawamura, M. Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3’-{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate reduction by xanthinexanthine oxidase. Anal. Biochem. 251(2), 206–209 (1997). 71. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72(1–2), 248–254 (1976). 72. Ohkawa, H., Ohishi, N. & Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358 (1979). Acknowledgements The authors acknowledge the Spanish Ministry of Science, Innovation and Universities and the European Union through projects DEP2014-58296-R, RTC-2017-6540-1, RTI2018-100934-B-I00, and FEDER program, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions L.M.L.T.: Methodology, formal analysis, investigation, writing-original draft. R.M.: Methodology, formal analysis, investigation, writing-original draft, visualization. G.K.: Methodology, formal analysis, investigation. M.G.: Methodology, formal analysis, investigation, visualization. P.A.: Conceptualization, funding acquisition, resources, methodology, visualization. J.M.P.: Conceptualization, supervision, project administration, resources, formal analysis, investigation, writing—review and editing. M.L.J.: Conceptualization, validation, formal analysis, supervision, writing—review and editing. Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to J.M.P. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. © The Author(s) 2021