cell biochemistry and function Cell Biochem Funct (2013) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3017

Leucine improves protein nutritional status and regulates hepatic lipid metabolism in calorie-restricted rats João Alfredo B. Pedroso1,2, Luciana Sigueta Nishimura1, Emídio Marques de Matos-Neto3, Jose Donato Jr.2 and Julio Tirapegui1* 1

Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil 3 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil 2

Several studies have highlighted the potential of leucine supplementation for the treatment of metabolic diseases including type 2 diabetes and obesity. Caloric restriction is a common approach to improve the health in diabetic and obese subjects. However, very few studies assessed the effects of leucine supplementation in calorie-restricted animals. Rats were subjected to a 30% calorie-restricted diet for 6 weeks to study the effects of leucine supplementation on protein status markers and lipid metabolism. Caloric restriction reduced the body weight. However, increased leucine intake preserved body lean mass and protein mass and improved protein anabolism as indicated by the increased circulating levels of albumin and insulin-like growth factor-1 (IGF-1), and the liver expression of albumin and IGF-1 messenger RNA. Leucine supplementation also increased the circulating levels of interleukin-6 and leptin but did not affect the tumour necrosis factor-α and monocyte chemotactic protein-1 concentrations. Ketone bodies were increased in rats consuming a leucine-rich diet, but we observed no changes in cholesterol or triglycerides concentrations. Caloric restriction reduced the liver expression of peroxisome proliferator activated receptor-α and glucose-6-phosphatase, whereas leucine supplementation increased the liver expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA) reductase and sterol regulatory element-binding transcription factor 1. A leucine-rich diet during caloric restriction preserved whole body protein mass and improved markers of protein anabolism. In addition, leucine modulated the hepatic lipid metabolism. These results indicate that increased leucine intake may be useful in preventing excessive protein waste in conditions of large weight loss. Copyright © 2013 John Wiley & Sons, Ltd. key words—branched-chain amino acids; interleukin-6; caloric restriction; IGF-1; albumin; supplementation

INTRODUCTION Amino acids are essential nutrients that serve as substrates for the synthesis of proteins and many other molecules. In addition, the carbon skeletons of the amino acids can be important sources of energy and glucose. Some amino acids also possess ergogenic properties, which makes them possible therapeutic nutritional supplements. Among all amino acids, the branched-chain amino acid leucine has received special attention because of its ability to stimulate protein synthesis and to affect metabolism.1,2 Cells have a leucine sensor that activates the mammalian target of rapamycin complex 1 (mTORC1)-signalling pathway as a function of leucine availability.3 mTORC1 acts as a key regulator of protein translation.4 Therefore, leucine represents an important nutritional signal to initiate protein synthesis through the activation of mTORC1 complex.5 Several studies have investigated whether leucine supplementation is able to improve protein balance during catabolic conditions. For instance, a leucine-supplemented *Correspondence to: Julio Tirapegui, Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, 580, bloco 14, São Paulo 05508-900, Brazil. E-mail: [email protected]

Copyright © 2013 John Wiley & Sons, Ltd.

diet preserved total carcass nitrogen and increased protein synthesis in skeletal muscle of rats bearing Walker 256 tumours, which is a condition resembling cachexia.6,7 In addition, postprandial stimulation of muscle protein synthesis in old rats was restored with a leucine-supplemented meal,8 suggesting that leucine-rich diets could be helpful in preventing muscle protein wasting during ageing.9,10 Leucine supplementation also improves the nitrogen balance and markers of protein nutritional status during conditions of protein malnutrition11,12 or food restriction13–15 In addition, leucine supplementation improves glucose and cholesterol metabolism in animals consuming a high-fat diet.16–19 Finally, several studies have highlighted the therapeutic potential of leucine supplementation for the prevention or treatment of metabolic diseases such as type 2 diabetes mellitus and obesity.20,21 However, despite the fact that caloric restriction is the most widely used nonpharmacological method to improve health in diabetic and obese subjects, very few studies have evaluated the consequences of leucine supplementation in calorie-restricted animals. Thus, the objective of the present study was to determine whether increased leucine intake has a positive impact on protein status and lipid metabolism markers in rats subjected to a 30% calorie-restricted diet. Received 6 June 2013 Revised 5 September 2013 Accepted 26 October 2013

j. a. b. pedroso et al. MATERIALS AND METHODS Animals Eight week-old male Sprague–Dawley rats were obtained from the Animal House of the Faculty of Pharmaceutical Sciences, University of São Paulo. The animals were maintained in individual cages under standard conditions of light (12-h light/dark cycle), temperature (22 ± 2 °C) and relative humidity (55 ± 10%). All animal procedures were approved by the Ethics Committee on Animal Experimental and were performed according to the ethical guidelines adopted by the Brazilian College of Animal Experimentation. Diets and experimental design The animals were distributed into three groups, which consumed distinct diets adapted from the AIN-93 M recommendations. The control group (n = 7) had unrestricted access to a diet supplemented with a mixture of non-essential amino acids (Table 1). The caloric restriction group (CR, n = 8) was subjected to a 30% caloric restriction and received a diet supplemented with a mixture of non-essential amino acids (Table 1). The caloric restriction + leucine supplementation group (CR + leu, n = 9) was subjected to a 30% caloric restriction and received a diet supplemented with leucine (Table 1). The diets in the calorie-restricted groups were adjusted to avoid any type of nutritional deficiency by increasing nutrients proportionally to the food restriction.22 Previous studies have suggested that leucine supplementation may produce a deficiency in valine and isoleucine.8 To prevent this possible deficiency, the diet of the CR + leu group was also supplemented with smaller amounts of these branched-chain amino acids (Table 1). We used the food intake of the control group as a reference to calculate the amount of food provided Table 1.

Composition of the experimental diets

Ingredients Starch Sucrose Casein Soy oil Cellulose Saline mix Vitamin mix L-Cystine Choline bitartrate Tetrabutyl hydroquinone Leucine Isoleucine Valine Alanine Aspartate Glycine Proline Serine kcal/g Lipids (%) Carbohydrate (%) Protein (%)

Control

RC

RC + leu

g/kg 566.84 100 140 40 50 35 10 1.8 2.5 0.008 — — — 9.27 13.85 7.812 11.982 10.937 3.8 9.45 70.01 20.54

g/kg 424.14 100 200 57.14 71.43 50 14.29 2.43 3.57 0.008 — — — 13.3 19.8 11.2 17.1 15.6 3.73 13.79 56.23 29.98

g/kg 404.71 100 200 57.14 71.43 50 14.29 2.43 3.57 0.008 71.43 15.71 9.29 — — — — — 3.73 13.79 54.15 32.06

Copyright © 2013 John Wiley & Sons, Ltd.

each day to the rats subjected to the caloric restriction (70% of the food consumption of the control group). In addition, the amount of food provided to each rat in the calorie-restricted groups was individualized for the specific animal in proportion to his body mass. These values were updated once per week to account for the changes in body weight (b.w.). The food was provided once per day, 8 h after the beginning of the dark period. Sampling and biochemical analysis At the end of the sixth week of supplementation, the rats were fasted for 8 h and were deeply anesthetized with a ketamine/xylazine cocktail. The animals were killed by decapitation, the blood was collected, and the liver was quickly dissected, frozen in liquid nitrogen and stored at 80 °C. Liver protein concentration was determined according to the method of Lowry et al.23 Total liver RNA was obtained through the RNA extraction using TRIzol reagent (Invitrogen), followed by the quantification using the Epoch Micro-Volume Spectrophotometer System (BioTek). Liver triglycerides content was extracted and calculated according to the method of Folch et al.24 Serum monocyte chemotactic protein-1 (MCP-1), leptin, interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and insulin-like growth factor-1 (IGF-1) were assessed using multiplex immunoassay through LINCOplex kits and an automated Lincoplex 200 instrument (LINCO Research). Serum total, high-density and low-density lipoprotein cholesterol, triglycerides and ketone bodies (acetoacetic and β-hydroxybutyric acids) were determined by enzymatic colorimetric methods using commercially available kits. Analysis of body composition The body composition of the animals (fat, protein and lean mass) was determined by chemical analysis of the carcass as previously described.6,13,25 The carcass comprised the whole body of the animal, except for the blood sample (approximately 5 ml) and the liver that were removed for tissue analysis. Initially, the whole carcass was dehydrated in a ventilated oven (approximately 70 °C) for 7 days. The whole dry carcass was then chopped and wrapped in gauze and filter paper for the determination of body fat by the solvent extraction technique using a Soxhlet apparatus and ethyl ether as solvent. The remaining carcass without humidity and fat was completely ground and sieved for the removal of hair which could reduce the homogeneity of the sample. This process resulted in a highly homogenous powder that was used to determine carcass protein by the microKjeldahl method. The amount of lean mass was calculated by subtracting absolute fat mass from total carcass mass. Fat, protein and lean mass contents were expressed in absolute terms (grams). Real-time polymerase chain reaction (qPCR) The RNA was extracted from the liver using TRIzol reagent, and 2 μg of total RNA was used for complementary DNA Cell Biochem Funct (2013)

effect of leucine supplementation in caloric restriction rats synthesis using random hexamers (Roche Diagnostics) and SuperScript II Reverse Transcriptase (Invitrogen). We used TaqMan PCR master mix and primers/probes (Applied Biosystems) for qPCR analysis [Albumin, Rn00592480_m1; IGF-1, Rn00710306_m1; sterol regulatory element-binding transcription factor 1 (SREBF1), Rn01495769_m1; peroxisome proliferator activated receptor-α (PPAR-α), Rn00566193_m1; HMG-CoA reductase, Rn00565598_m1; glucose-6-phosphatase, Rn00689876_m1]. The relative quantification of messenger RNA (mRNA) was calculated by the 2 ΔΔCt method using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression as the normalizing factor. GAPDH expression was determined using a SYBR Green master mix (forward: 5’- ccgttcaggtctgggatgac-3’ and reverse: 5’-gggcagcccagaacatcat-3’). The data are reported as fold changes compared with values obtained from control group. The assays were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems).

Statistical analysis One-way analysis of variance (ANOVA) was used for comparison between groups, and differences were evaluated using the Newman–Keuls test. Partial correlations between the data were determined using linear regression. An alpha value of 0.05 was considered to be significant for all analyses. The results are expressed as the mean ± standard error of the mean (SEM). The statistical analysis was performed using the GraphPad Prism software.

RESULTS Leucine supplementation preserved lean mass and improved markers of protein anabolism in calorie-restricted rats The groups began the experiment with similar b.w. (P = 0.53, Figure 1A). The rats in the CR and CR + leu groups consumed all feed offered. The average food intake of CR and CR + leu groups was about 30% less compared with control group (P < 0.001). The average food intake was similar between CR and CR + leu groups [control: 34.5 ± 0.9 g/100 g b.w.; CR: 24.4 ± 0.1 g/100 g b.w.; and CR + leu: 24.5 ± 0.1 g/100 g b.w.]. As expected, the calorierestriction significantly decreased the b.w. of CR and CR + leu groups. However, the rats in the CR + leu group retained a higher b.w. after 5 and 6 weeks of caloric restriction compared with those of the CR group (P < 0.05, Figure 1A). After 6 weeks of caloric restriction, the rats in the CR group lost 16.5 ± 2.2% of their initial b.w. compared with a gain of 19.9 ± 2.4% in the control group (P < 0.0001, Figure 1A). In contrast, the rats in the CR + leu group lost only 8.3 ± 1.3% of their initial b.w. (P < 0.01 versus CR group). The results of the body composition assessment showed that that caloric restriction significantly reduced the body lean mass, body protein content and body fat mass (Figure 1B). However, the rats in the CR + leu group showed a partial preservation of all components of the body composition. Next, we assessed markers of protein nutritional status, including, protein and total RNA concentrations in the liver, and albumin and IGF-1 levels. CR group showed a decreased concentration of protein and total RNA in the liver compared

Figure 1. Leucine supplementation preserved lean mass and improved markers of protein anabolism in calorie-restricted rats. Body weight (A), body composition (B), serum concentration of insulin-like growth factor-1 (IGF-1) (C), liver IGF-1 messenger RNA (mRNA) expression (D), serum concentration of albumin (E) and hepatic albumin mRNA expression (F) of control, CR and CR + leu groups. *: significantly different (P < 0.05) from the CR group. Superscripts letters: conditions without a common letter are significantly different (P < 0.05) Copyright © 2013 John Wiley & Sons, Ltd.

Cell Biochem Funct (2013)

j. a. b. pedroso et al. with control group (Table 2). However, leucine supplementation partially preserved these nutritional markers (Table 2). Serum IGF1 was decreased in the rats of the CR group but partially preserved in the rats of the CR + leu group compared with those of the control group (Figure 1C). Consistent with this effect, we observed decreased IGF-1 mRNA expression in the liver of CR group compared with control rats. Remarkably, leucine supplementation preserved liver IGF-1 mRNA expression to levels comparable with the control group (Figure 1D). Serum albumin concentrations and liver albumin mRNA expression were also decreased in the rats of the CR group in comparison with the values found in the control group. Leucine supplementation completely prevented the decreases in serum albumin and liver albumin mRNA expression (Figure 1E–F). Increased concentrations of interleukin-6 in calorie-restricted rats receiving leucine supplementation Recent studies have found that leucine supplementation can act on multiple levels of metabolism, which influence glucose and lipid metabolism in animals with diet-induced obesity.16,18 However, much less is known about the effects of leucine under conditions of caloric restriction. We analysed the serum concentrations of several pro-inflammatory cytokines: leptin, MCP-1, TNF-α and IL-6. We observed a decrease in the serum leptin concentration in the rats of the CR and CR + leu groups compared with the control animals (Table 3). However, the rats of the CR + leu group showed higher leptin levels than those of the CR group (P < 0.05). The leptin levels showed a positive correlation with body fat mass (P < 0.0001, r = 0.974). MCP-1 levels were decreased in the rats of the CR and CR + leu groups compared with those of the control group (Table 3). We observed no significant differences in the TNF-α levels among the groups. In contrast, the levels of IL-6 in the rats

Table 2.

of the CR + leu group were significantly higher than those of the CR group (Table 3). Leucine supplementation increased the levels of ketone bodies and affected the hepatic expression of genes involved in lipid metabolism Our previous results and those of other studies15,26–28 have found that the liver is an important target of leucine. Thus, we assessed the serum lipid profile and the liver expression of key genes involved in the regulation of metabolism. Total cholesterol and high-density lipoprotein cholesterol were significantly higher in calorie-restricted rats, independent of the leucine supplementation (Figure 2A). We found a trend (P = 0.05) towards an increase in low-density lipoprotein cholesterol in the rats of the CR and CR + leu groups compared with those of the control group. The triglyceride levels did not differ significantly among the groups (Figure 2A). We also assessed the levels of circulating ketone bodies and found a reduction in the rats of the CR group compared with the control rats. However, the levels of ketone bodies in the rats of the CR + leu group were similar to those of the control rats and significantly higher than those of the CR group (Figure 2A). CR and CR + leu groups showed a similar reduction in liver triglycerides content compared with control group (Figure 2B). We also observed a reduction in the mRNA expression of PPAR-α and glucose-6-phosphatase in the livers of the CR and CR + leu group rats compared with the control group (Figure 2C). HMG-CoA reductase mRNA expression was higher in the rats of the CR group than in the control rats and was further increased in the CR + leu group rats. SREBF1 mRNA expression was not different between the CR group rats and the control animals but was increased in the rats of the CR + leu group in comparison with the other groups (Figure 2C).

Protein and total RNA concentrations in the liver Control

CR a

Protein(mg/100 mg tissue) Total RNA(μg/mg tissue)

12.65 ± 0.33 a 1.47 ± 0.01

CR + leu b

11.61 ± 0.28 b 1.38 ± 0.01

P ab

11.98 ± 0.20 a 1.45 ± 0.01

0.0454

Leucine improves protein nutritional status and regulates hepatic lipid metabolism in calorie-restricted rats.

Several studies have highlighted the potential of leucine supplementation for the treatment of metabolic diseases including type 2 diabetes and obesit...
308KB Sizes 0 Downloads 0 Views