Leucine‐rich diet minimises liver glycogen mobilisation and modulates liver gluconeogenesis enzyme expression in tumour‐bearing cachectic rats

Cachexia is defined as a complex metabolic syndrome that is associated with tissue damage. Some studies have shown that the liver metabolic alterations contribute to overall host tissue wasting. Knowing that leucine acts as cell signalling, we evaluated hepatic metabolism in Walker 256 tumour‐bearing rats and investigated the modulatory effects of a leucine‐rich diet.


Introduction
Cachexia is a multifactorial syndrome that significantly affects patient quality of life and is responsible for nearly to 20-30% of all cancer-related deaths [1,2]. Cancer cachexia induces metabolic alterations that include primarily disorders of protein turnover, leading to significant skeletal muscle wasting, with or without fat loss, and subsequent involuntary body weight loss [3]. Cachexia leads to chronic inflammation, which elevates circulating levels of Creactive protein (CRP) and pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumour necrosis factoralpha (TNF-α) [4], which may play an important role in the mechanisms that underlie muscle wasting [5].
Moreover, a study performed by Narsale and colleagues [4] showed that chronic exposure to IL-6 induces hyperplasia in the hepatic tissue. This liver hypertrophy may contribute to cachexia progression in a cachectic mouse model by elevating resting energy expenditures [4]. The liver tissue is partly responsible for the systemic metabolic rate by regulating pathways involved in the utilisation, transport, storage and breakdown of glucose and fat. These important liver functions could be jeopardised in the presence of cancer, leading to the abnormal expression of liver enzymes, which leads to a poor prognosis in most cancers [6].
The majority of neoplastic cells preferentially use glucose as an energy source, resulting in increased glucose metabolism in cancer cells [7]. In cancer patients who are losing weight, hepatic gluconeogenesis is enhanced [8], probably to provide the tumour cells with de novo synthesised glucose, which is produced by using amino acids from skeletal muscle breakdown, glycerol from lipolysis and lactate from tumour aerobic glycolysis (Warburg effect) [9]. The gluconeogenesis process depends on the adenosine triphosphate (ATP) supply, which leads to energy depletion under low serum glucose conditions, contributing to protein breakdown, followed by tissue wasting and weight loss in cancer patients [10]. If we consider skeletal muscle tissue wasting to be the main source of amino acids for liver gluconeogenesis, it is plausible to assume that energy depletion contributes to skeletal muscle loss, which in turn causes the cachectic state [11,12].
Several studies are currently investigating strategies to inhibit or minimise skeletal muscle wasting and consequently improve quality of life for cancer patients [13][14][15]. One of these strategies is nutritional supplementation with leucine, which is a branched-chain amino acid (BCAA) that plays an important role in skeletal muscle metabolism [16]. Leucine efficiently improves skeletal muscle protein synthesis in patients [17,18] and in animals, as shown previously [19][20][21][22]. Leucine alone and in a complete BCAA mixture stimulates protein synthesis and decreases protein proteolysis [23][24][25]. Leucine regulates protein synthesis in the skeletal muscle following food intake, stimulating the mTOR pathway and inhibiting the ubiquitinproteasome pathway [23,26]. Moreover, a recent study performed by Takegoshi and colleagues [25] revealed that oral BCAA supplementation in patients with liver cirrhosis potentially suppresses the incidence of hepatocellular carcinoma, inducing anti-fibrotic factors and preventing apoptosis.
Therefore, this study aimed to determine if a leucine-rich diet could modulate the liver damage caused by tumour growth, improving hepatic enzymes and restoring liver glycogen stores, even in the cachectic state.

Animals and diet
Female Wistar rats (approximately 90±10 days old, obtained from the Animal Facilities at the State University of Campinas, UNICAMP, Brazil) that weighed approximately 265±10 g were housed in collective cages under controlled environmental conditions (light and dark 12/12 hr; temperature 22±2°C; and humidity 50-60%). The animals were monitored daily, weighed 3 times/week and given food and water ad libitum. Semipurified diets were constructed in accordance with the American Institute of Nutrition (AIN-93; [27]), and the leucine-supplemented diet was enriched with 3% L-Leucine, as described in our previous works [19,[28][29][30]. Both diets (control (C) and leucine (L)) contained similar amounts of nitrogen (approximately 2.84 g N 2 /100 g diet), for a protein content of approximately 18%.

Walker 256 tumour inoculation
The Walker 256 tumour is widely used as an experimental model of cancer cachexia syndrome [19,[30][31][32]. Walker 256 carcinosarcoma cells (2.5 × 10 6 viable cells) were injected subcutaneously into the right flank of the experimental rats on the first day of the experiment. The general guidelines of the UKCCCR (United Kingdom Co-ordinating Committee on Cancer Research, 1998) [33] regarding animal welfare were followed, and the experimental protocol was approved by the Institutional Committee for Ethics in Animal Research (CEEA/IB/UNICAMP, protocol # 2677-1).

Experimental protocol
The animals were randomly distributed into four experimental groups according to tumour implantation status and leucine nutritional supplementation. Two groups were fed a control diet (18% protein): C, control group (n=9) and W, Walker 256 tumour-bearing group (n=9). The other two other groups were fed a leucinerich diet (18% protein + 3% leucine): L, leucine control group (n=8) and LW, leucine Walker 256 tumour-bearing group (n=9). All rats were monitored and weighed 3 times/week. At the end of the nutritional supplementation period (i.e., 21 days after tumour evolution), the animals were euthanised without an overnight fast, and blood, gastrocnemius muscle and liver samples were collected. The collected tissues were resected and weighed, and some liver fragments were immediately frozen directly in liquid nitrogen and stored at −80°C for biochemical analyses, western blotting and real-time PCR. Additional fragments of liver tissue were immediately fixed in 4% paraformaldehyde for 24 hours before being processed for histology with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. The blood samples were centrifuged at 10,000 × g at 4°C for 10 min, and serum was stored at −20°C for biochemical analyses.

Biochemical analyses
Serum was analysed to determine the total protein, glucose and bilirubin contents, as well as some enzyme activities, such as alkaline phosphatase (ALP), gamma-glutamyltransferase (γGT), glutamate pyruvate transaminase (GPT), and glutamate oxaloacetate transaminase (GOT). ALP, γGT, GPT, GOT and cholesterol were also measured in the liver tissue. The serum total protein content was determined using the Biuret reactive reagent, and the absorbance was read at 540 nm. The glucose levels were measured using the glucose-oxidase reactive reagent, and the absorbance was read at 540 nm. Serum ALP activity was measured using a P-NPP substrate solution (60 mM nitrophenyl phosphate (P-NPP), 10 mM MgCl 2 6H 2 O, pH 10.3). The enzymatic reaction proceeded for 30 min at 37°C, and the absorbance was read at 405 nm. γGT levels were measured using an enzymatic assay with a γGT substrate (g-carboxyl-4-nitroanilide), and the absorbance was read at 405 nm within 10 minutes. The GPT and GOT levels in the serum and liver tissue were measured using an enzymatic assay kit (Bioclin, Sao Paulo, Brazil), with a GPT and GOT substrate (L-Alanine + α-ketoglutarate; L-Aspartate + α-ketoglutarate), with the enzymatic catalytic reactions performed according to manufacturer instructions. The absorbance was read at 365 nm using a microplate reader (Hidex Chameleon TM V plate reader, Switzerland). Cholesterol levels were measured using an enzymatic assay with a substrate (4-aminophenazone, phenol, cholesterol oxidase, cholesterol esterase and peroxidase). The reaction was performed according to the manufacturer's instructions (Laborclin), and the absorbance was read at 500 nm (Hidex Chameleon TM V plate reader, Switzerland).

Histology
The fixed liver tissues were embedded in paraffin, cut into 5 μm sections and stained with H&E and PAS. H&E-stained slides were analysed to determine the hepatocyte area, the nuclear hepatocyte area and the number of hepatocyte nuclei. These analyses were achieved by counting 5 fields (500 μm 2 each) on one slide from each animal, with at least six rats per group. The images were captured using a Leica DMLS microscope at a magnification of 200X. PAS staining was analysed to determine the liver glycogen content. The Image-Pro Premier 9.1.4 software was used to measure the glycogen content, using the tool Line Profile, which represents the pixel intensity value as a line drawn on the image. Thus, higher intensity pixels indicate higher glycogen content.

Western blotting
Liver tissue samples were homogenised in protein extraction buffer (20 mM N-2-hydroxy ethylpiperazine-N-2-ethanesulfonic acid, 100 mM KCl, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 1 mM DAB tetrahydrochloride, 0.5 mM orthovanadate and 50 mM glycine, pH 7.4), followed by centrifugation at 10,000×g for 15 min at 4°C. The supernatant samples were then separated by 12.5% SDS-PAGE electrophoresis under reducing conditions. After gel electrophoresis and protein transfer onto a nitrocellulose membrane, the proteins were blocked in 5% non-fat dry milk at room temperature for 1 h. The membranes were then incubated overnight at 4°C with antibodies against total AMPK (Catalogue number: 2603S; Cell Signalling; diluted 1:1,000) and phospho-AMPK (Thr172) (Catalogue number: 2535S; Cell Signalling; diluted 1:1,000). Immunoreactivity was detected via the sequential incubation of the membranes with an antimouse secondary antibody for 1 h at room temperature, which was visualised using a chemiluminescence detection system. The level of AMPK was estimated relative to the level of the 37 kDa GAPDH protein (Catalogue number: 5174; Cell Signalling; diluted 1:1,000).

Statistical analyses
The results are shown as the mean ± standard deviation, and all data were analysed using the Graph Pad Prism 6.0 software (Graph-Pad Software, Inc.). For comparisons among multiple groups (e.g., C, W, L and LW), the data were evaluated with analysis of variance (two-factor ANOVA) followed by the post-hoc Bonferroni's test [34].

Morphological and histological parameters
Walker-256 tumour growth led to cachexia, as indicated by significant weight loss, represented by the carcass weight, in both tumour-bearing groups (W and LW). On the other hand, skeletal muscle mass loss was found only in the W group, and we observed no differences between the LW and L groups. The relative tumour weight ratios were similar in groups W and LW. The relative liver weight was greater in groups W and LW than in groups C and L (Table 1).
Walker-256 tumour growth leads to some alterations in liver morphology. The higher liver weight in the W and LW groups (Table 1) was associated with a greater number of hepatocyte nuclei, as well as a greater nuclear hepatocyte area, in both tumour-bearing groups, with statistical significance only for W rats. The leucinerich diet scheme led to changes in these parameters in the L group in comparison to the C group. The liver glycogen content was lower in the W group than in the control group (C). However, the glycogen content was preserved in the LW group, with no difference between the LW and L groups (Figure 1). Data are expressed as the mean ± SD. Legend: C control; W, tumour-bearing (fed a control diet, 18% protein); L, control; LW, tumour-bearing (fed a leucine-rich diet, 18% protein + 3% leucine). Relative tissue weights correspond to the ratio of the specific tissue and body weights, expressed as a percentage. For details, please see the Material and Methods section. a P < 0.05 in comparison with the C group; b P < 0.05 in comparison with the W group and c P < 0.05 in comparison with the L group.

Serum and liver tissue biochemical analyses
Both tumour-bearing groups had significantly lower total serum protein concentrations (W and LW) than the control groups ( Table 2). The glucose level was lower in the W group than in the control group (C), but no difference was observed between the LW and L groups ( Table 2). The serum bilirubin concentration was greater in both tumour-bearing groups (W and LW). Additionally, serum ALP activity was higher in groups W and LW than in their respective control groups, and the serum γGT content was greater in the tumour-bearing groups, but no differences in GPT and GOT content were observed, independent of tumour status or nutritional scheme ( Table 2).
The liver tissue analysis showed that hepatic enzyme activities, such as γGT, GOT and GPT, had the same values among the groups (Table 2). However, liver alkaline phosphatase activity was higher in both tumourbearing groups. In addition, while the liver cholesterol  (Table 2).

Molecular analyses
Glycogen synthase, phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDHA) gene expression levels were higher in the W group than in the other groups. Therefore, these enzyme expression levels were unchanged in group LW in comparison to the respective control group (L). In addition, these genes were expressed at significantly lower levels in group LW than in group W ( Figure 3B-C). On the other hand, glucokinase and citrate synthase gene expression did not vary among the experimental groups ( Figure 3A and E). However, AMP-activated protein kinase (AMPK) activation was higher in both tumour-bearing groups than in the control groups ( Figure  3F). Data are expressed as the mean ± SD. Legend: C control; W, tumour-bearing (fed a control diet, 18% protein); L, control; LW, tumour-bearing (fed a leucine-rich diet, 18% protein + 3% leucine). For details, please see the Material and Methods section a P < 0.05 in comparison with the C group, b P < 0.05 in comparison with the W group and c P < 0.05 in comparison with the L group.  Illustration of the proposed mechanism by which a leucine-rich diet improves cachexia in Walker 256 tumour-bearing rats. TCA: tricarboxylic acid cycle. Dashed arrows and lines represent less stimulated pathways, with the hypothesis supported by the effects of leucine. Solid thicker lines and arrows represent more stimulated pathways, seen in the tumour-bearing group (W).

Discussion
Cachexia is responsible for 20-30% of all cancerrelated deaths and also leads to a poor quality of life in these patients. Thus, understanding the mechanisms involved in cancer cachexia and the effects of coadjuvant treatments is essential. In this study, we investigated the damage caused by Walker 256 tumour growth in the liver tissue and also showed the effects of a leucine-rich diet on the liver tissue in these cachectic rats.
Cachexia leads to significant skeletal muscle loss. On the other hand, some visceral organs, such as the heart, spleen, and liver, maintain their mass or even exhibit hypertrophy during cachexia [4]. Corroborating the literature, the increased liver mass that was found here in both tumour-bearing groups likely reflected the possibly related chronic inflammatory state that is present in cachexia hosts, which is caused primarily by an increase in circulating pro-inflammatory cytokines, such as IL-6, IL-1 and TNF-α [28]. Some studies reported that chronic exposure to IL-6 induces hyperplasia in hepatic tissue [4,35]. In our previous studies, we found that serum levels of pro-inflammatory cytokines, especially IL-6 and TNF-α, were higher in Walker 256 tumour-bearing rats [36] and in MAC 16 tumour-bearing mice (another widely used experimental cancer cachexia model) [28].
Liver hypertrophy is speculated to contribute to cachexia progression in cancer patients by elevating resting energy expenditures [37]. The liver tissue is partly responsible for controlling the systemic metabolic rate by regulating a variety of pathways involved in the transport, storage, breakdown and utilisation of glucose. In these processes, some liver glycolytic enzymes could be changed during tumour evolution, as we have observed here, especially in the W group. The stronger expression of glycogen synthase in tumour-bearing rats in this study was not consistent with reduced glycogen stores. This observation could be explained by the fact that the glycogen produced by glycogen synthase in the liver might be immediately mobilised to provide glucose to meet the growing neoplastic tissue demands. Although enhanced liver lactate dehydrogenase expression could be related to greater lactate content (observed in our previous study [14]), this fact likely suggests higher liver gluconeogenesis.
Cancer cells preferentially use glucose as an energy source, leading to an increased glycolysis rate in tumour tissues and a consequent reduction of the blood glucose concentration [38], even with higher liver gluconeogenesis. Additionally, some cancer cells exhibit disturbed glucose metabolism, known as the Warburg effect, in which the cells prefer glycolysis over oxidative phosphorylation, even in the presence of oxygen [39]. In addition, liver gluconeogenesis is enhanced in cachexia because the gluconeogenic precursors (amino acids from proteolysis, glycerol from lipolysis and lactate from glycolysis) are converted to de novo glucose that probably feeds the tumour (this process is called a "futile energy cycle" and was first described by Cori and Cori [40]). Since the Cori cycle is maintained by intense phosphoenolpyruvate carboxykinase (PEPCK) activity, the higher PEPCK gene expression observed here likely represented an enhanced gluconeogenesis process in the W group, which could lead to greater energy expenditures and more muscle wasting, as observed in the present study. Conversely, liver PEPCK expression exhibited no differences between the L and LW groups. This result could probably be explained by the fact that tumour tissues might use leucine directly as an energy source. Using leucine as an energy source, the tumour needs less glucose; consequently, the gluconeogenesis process is less important, and less energy is spent, reflecting the preserved skeletal muscle tissue in the LW group. In our recent study [30], which used the same experimental protocol used in this study, we found a lower cachexia index in Walker 256 tumour-bearing rats that were fed a leucine-rich diet. This outcome is likely related to the reduced gluconeogenesis that was observed in these animals supplemented with leucine. In this same study [30], we found higher lactate levels (19.2%) in the W group than in the C group, which were significantly lower (15.3% lower; [27]) in tumour-bearing rats fed a leucine rich-diet (LW) [30]. This result suggests that leucine supplementation reduced tumour lactate release and also decreased skeletal muscle wasting, leading to lower amino acid levels. Consequently, the liver receives less lactate and amino acids, leading to a lower gluconeogenesis rate in the LW group. Reduced gluconeogenesis results in reduced energy expenditures, which can contribute to the lower cachexia index that was previously observed in tumour-bearing rats fed a leucine-rich diet [30] (Figure 3).
Although a leucine-rich diet benefited some aspects of energy metabolism, the activation of hepatic AMPK (activated under conditions of nutrient stress [41]) was higher in both tumour groups, suggesting a reduced energy supply. Thus, despite having lower similar blood glucose levels (LW = W), corroborating greater AMPK activation, the hepatic glycogen was mobilised in the W group but preserved in leucine-treated Walker 256tumour-bearing rats. This fact suggested the partially modulatory effect of leucine nutritional supplementation. Although we evaluated some liver parameters, such as bilirubin, and serum enzymes, such as alkaline phosphatase (ALP) and gammaglutamyltransferase (γGT), that are associated with liver damage in cancer patients and tumour-bearing rats, a leucine rich-diet could not prevent changes in these parameters. Serum ALP activity provides a sensitive indicator of obstructive and space-occupying lesions of the liver [42]. Therefore, leucine was not able to prevent this kind of liver damage. On the other hand, the leucine-supplemented tumour group had the same γGT levels as the W group. As an oxidative stress marker, γGT can give rise to pro-oxidant reactions that can produce endogenous reactive oxygen species (ROS) in tumour cells and play a major role in tumour formation, cell proliferation and apoptosis [43]. Thus, the leucine richdiet had no effect on the oxidative stress observed in tumour-bearing hosts. While leucine was not effective in protecting against the elevation of these parameters that leads to impaired liver function, leucine treatment resulted in some positive effects, such as the maintenance of skeletal muscle mass, glycogen stores and normal levels of genes related to gluconeogenesis, as shown in Figure 3.
Because the liver has the potential to contribute to several wasting-associated mechanisms, further studies are now underway in our laboratory to understand the role of the liver in cancer cachexia progression and investigate how the effects of coadjuvant treatments can benefit this tissue.

Conclusion
Our results suggest that during Walker 256 tumour growth, a leucine-rich diet is effective in protecting against host skeletal muscle tissue wasting and leads to benefits in the liver tissue. The leucine-rich diet induces no protection against the elevation of some hepatic damage markers but maintains energy stores and likely decreases the futile Cori cycle in the liver, reducing energy expenditures in tumour-bearing rats.