Effects of legume protein hydrolyzates on lipid metabolism in an obese rat experimental model. Interaction with aerobic physical exercise/efectos de hidrolizados proteicos vegetales procedentes de leguminosas sobre el metabolismo lipídico en un modelo experimental de rata obesa. Interacción con el ejercicio físico aeróbico

Supervised by:
  1. Pilar Aranda Ramírez Director
  2. Jesus Maria Porres Foulquie Director

Defence university: Universidad de Granada

Fecha de defensa: 17 February 2015

  1. Juan Llopis González Chair
  2. José Ángel Rufián Henares Secretary
  3. Juana M. Frías Arevalillo Committee member
  4. Goua Marie Committee member
  5. Antonio Martínez-Amat Committee member

Type: Thesis


Metabolic syndrome (MetS) is a cluster of interrelated metabolic conditions which increase the risk of developing cardiovascular disease (Kaur, 2014). MetS is characterized by central obesity, dyslipidemia, elevated blood pressure, and elevated plasma glucose (Gonçalves et al., 2014). Patients with MetS are also more susceptible to develop type 2 diabetes mellitus (Reaven, 2004), and their hepatic morphology and function can be adversely affected leading to the development of Non Alcoholic Fatty Liver Disease (NAFLD) (Marchesini et al., 2003). This specific pathology is characterized by steatosis, lobular and portal inflammation, hepatocyte ballooning, and fibrosis (Brunt & Tiniakos, 2010). Furthermore, NAFLD pathology is now considered as the liver manifestation of MetS (Angelico et al., 2005). Although the exact mechanisms leading to it are not yet completely understood, insulin resistance and chronic oxidative stress have been reported to play a major role in liver damage and development of NAFLD (Polyzos, Kountouras, & Zavos, 2009; Rolo, Teodoro, & Palmeira, 2012). The study of MetS and the development of strategies for its prevention and treatment has attracted increasing attention in recent years due to its growing prevalence and associated comorbidities exemplified by cardiovascular disease and NAFLD (Kaur, 2014; Marchesini et al., 2003). Changes in lifestyle, i.e. caloric restriction, low fat and low glycemic index diets, consumption of foods rich in beneficial bioactive ingredients, and regular physical activity, are the primary interventions chosen to improve the alterations associated with the specific pathology. Legumes represent an excellent source of essential nutrients and exhibit a variety of health effects related to their antioxidant (Doblado, Frías, & Vidal-Valverde, 2007) and hypolipidemic properties (Hermsdorff et al., 2011). Such important properties derive from the specific characteristics of legume proteins, carbohydrates and lipids, and also from several non-nutritional compounds like polyphenols, phytic acid or ¿-galactoside oligosaccharides. Biotechnological treatments applied to legumes not only improve their nutritional value (Porres et al., 2003), but may also enhance their health-promoting potential (Doblado et al., 2007). The nutritional relevance of lupin (Lupinus spp.) has gained increased attention due to its high content of protein, minerals, dietary fibre, and fat (Porres et al., 2006), as well as to its low levels of non-nutritional components such as trypsin inhibitors, lectins, or alkaloids in the sweet varieties. In addition to these nutritional properties, lupin also features beneficial functional properties such as antioxidant or hypocholesterolemic effects (Martínez-Villaluenga et al., 2009; Parolini et al., 2012). Vigna radiata is another widely used legume for human diets. Its interesting nutrient and bioactive compound composition (Dahiya et al., 2013; Tang, Dong, Guo, Li, & Ren, 2014) can be significantly improved by technological processing like sprouting (Fernandez-Orozco et al., 2008; Silva et al., 2013). In fact, V. radiata sprouts are usually commercialized to take part in numerous healthy dishes that are becoming increasingly popular for the general consumer. In recent years, new processing conditions are continuously emerging in order to improve both the nutritional and health-related properties of legume-derived foodstuffs. One good example of such new products is protein hydrolyzates, which can be produced using different chemical and biological techniques. Among the most widely used methodologies to prepare protein hydrolyzates is protein extraction under alkaline conditions followed by hydrolysis process using different exoproteases like alcalase or flavourzyme (Megías et al., 2007). However, other alternative biotechnological treatments like germination or fermentation also render protein preparations with important degree of hydrolysis and the presence of bioactive compounds with beneficial health actions (Porres et al. 2003; Urbano et al. 2003; Kapravelou et al. 2013). Via such treatments and in particular the germination process, enhancements of protein contents and antioxidant activity of the legumes can be achieved (Doblado et al., 2007; Ghavidel & Prakash, 2007). As a result, the resulting bioactive peptides may act as potential physiological modulators of metabolism, given that they inhibit the activity of angiotensin converting enzyme and exhibit antioxidant and bile acid-binding properties (Yoshie-Stark & Wäsche, 2004), thus showing promising potential as functional ingredients. Changes in lifestyle habits have been suggested as a valuable strategy for the combined treatment of MetS. In this context, the effects of different types of exercise have been further studied. High-intensity aerobic interval training (HIIT) has been reported to be more effective at reducing cardiovascular disease risk in rats with metabolic syndrome than moderate-intensity continuous training (Haram et al., 2009). With regard to liver metabolism, several authors have studied the effect of moderate or vigorous intensity exercise on different aspects of NAFLD. Moderate intensity cycling exercise during four weeks significantly reduced visceral adipose tissue, hepatic triglyceride concentration, and plasma free fatty acids without altering body weight (Johnson et al., 2009). Moreover, moderate intensity exercise training showed beneficial effects on intrahepatic triglyceride content, although it did not improve hepatic lipoprotein kinetics in obese individuals with NAFLD (Sullivan et al., 2012). Different in vivo experimental models have been used to test the beneficial effects of diet and exercise on several parameters of MetS. One of the most extended methods to test the hypolipidemic properties of foodstuffs is the consumption by rats of a diet rich in cholesterol and/or saturated fat with strong atherogenic index, based on that originally formulated and assayed by (Nath et al., 1959). In such an experimental model, the potential hypolipidemic effect of the tested compound or mixture can be studied at both the digestive or metabolic level. Another interesting animal experimental model that is widely accepted as effective tool to study the multifactorial effects of diet and exercise on MetS associated conditions is the obese Zucker rat, which is known to present a genetic defect in leptin receptor that causes the development of hyperphagia leading to obese phenotype (Galisteo, Duarte, & Zarzuelo, 2008). In addition to obesity, the obese Zucker rat model shares many similarities with humans affected by MetS, including dyslipidaemia, insulin resistance, hepatomegalia, altered antioxidant status, and inflammatory process (Galisteo et al., 2010; Hey-Mogensen et al., 2012). The main objective of this Doctoral research work was to test the beneficial effects of different healthy lifestyle strategies, including nutrition and physical exercise, on several parameters of metabolic syndrome. In order to do so, the present Doctoral Thesis was structured in two different and complementary phases with their corresponding objectives: Phase 1: Effects of Lupinus albus protein hydrolyzate and insluble dietary fiber residue in an experimental model of diet-induced hypercholesterolemia In the first set of experiments, we aimed to study the potential of lupin protein hydrolyzate, combined or not with lupin insoluble fiber, as functional food ingredients with beneficial effects on different metabolic parameters using an in vivo experimental model of rats fed a diet rich in cholesterol and coconut oil to test: 1) their hypolipidemic action, and 2) their influence on certain parameters of hepatic, renal and large intestine functionality. Phase 2: Effects of raw and 4d-germinated Vigna radiata flours combined with a HIIT protocol in an experimental model of genetically obese Zucker rat In the second set of experiments, we aimed to assess the effects of a high intensity aerobic intervallic training protocol combined with the consumption of raw and 4-day-germinated V. radiata on glucose and lipid metabolism parameters, liver histology and functionality, and hepatic antioxidant status in an animal experimental model of metabolic syndrome, the obese Zucker rat, that presents hepatic alteration related to NAFLD. In phase 1 experiments, rats were fed hypercholesterolemic high-fat diets in which different protein (casein vs lupin protein hydrolyzate) or insoluble dietary fiber (cellulose vs lupin insoluble fiber residue) sources were included to test the potential benefits of lupin products on the experimental animal model of diet-induced hypercholesterolemia/ hyperlipidemia. The experimental model developed exhibited significant alterations in plasmatic total-cholesterol and triglycerides, morphological and compositional changes in the liver, and alterations in urinary parameters of renal function. Dietary inclusion of lupin protein hydrolyzate and insoluble fiber residue caused a significant decrease in plasma and hepatic triglyceride content, and appeared to improve glucose metabolism. Furthermore, lupin functional ingredients induced diverse potentially beneficial changes in kidney, liver, and large intestine functionality, although they could not reverse the histological alterations found in liver. In phase 2, experiments were designed to test the influence of a HIIT protocol consisting of successive 4 min periods at 65-80% of VO2max, followed by 3 min recovery periods at 50-65% of VO2max, combined with the consumption of diets containing 70% of their total protein content and nearly 100% of their total dietary fiber content supplied as raw or 4-day-germinated V. radiata, on plasma and liver biochemical parameters of obese and lean Zucker rats. Obese rats exhibited higher food intake and body weight, and suffered significant alterations in plasma lipid profile, Area under the Curve after oral glucose overload, liver histology and functionality, and antioxidant status. Exercise increased the aerobic capacity of both rat phenotypes and diminished the severity of metabolic syndrome alterations, especially those related to glucose and lipid metabolism, affecting the levels and activity of proteins involved in metabolic pathways and the gene expression of GPX4, a key antioxidant enzyme, in the liver. Consumption of germinated V.radiata was efficient at reducing body weight, hepatomegalia and hepatic triglyceride content of obese Zucker rats, as well as improving their liver functionality and antioxidant status. The combination of both lifestyle strategies (plant protein hydrolyzates and physical exercise) had superior effects when compared to the combination of animal protein and physical exercise and could represent an efficient and feasible complement to the more severe pharmacological treatments that are currently in use. Therefore, such lifestyle strategies could contribute to lowering the dosage of such treatments in patients that suffer alterations caused by the MetS.