Diabetogenic effect of pravastatin is associated with insulin resistance and myotoxicity in hypercholesterolemic mice.

BACKGROUND: HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs widely used to treat hypercholesterolemia and prevent cardiovascular disease. Statins are generally well tolerated, but adverse reactions may occur, particularly myopathy and new onset of diabetes. The exact mechanism of statin-induced myopathy and diabetes has not been fully elucidated. We have previously shown that treatment of hypercholesterolemic (LDLr-/-) mice with pravastatin for 2 months decreased pancreatic islet insulin secretion and increased oxidative stress and cell death, but no glucose intolerance was observed. The purpose of the current work was to study long-term pravastatin effects on glucose homeostasis, insulin sensitivity, muscle protein turnover and cell viability. METHODS: LDLr-/- mice were treated with pravastatin for 3, 6 and 10 months. Glucose tolerance, insulin resistance and glucose-stimulated insulin secretion were evaluated. The rates of protein synthesis and degradation were determined in gastrocnemius muscle after 10 months of treatment. Insulin signalling, oxidative stress and cell death were analysed in vitro using C2C12 myotubes. RESULTS: After 6 and 10 months of treatment, these mice became glucose intolerant, and after 10 months, they exhibited marked insulin resistance. Reduced islet glucose-stimulated insulin secretion was observed after the 3rd month of treatment. Mice treated for 10 months showed significantly decreased body weight and increased muscle protein degradation. In addition, muscle chymotrypsin-like proteasomal activity and lysosomal cathepsin were markedly elevated. C2C12 myotubes exposed to increasing concentrations of pravastatin presented dose-dependent impairment of insulin-induced Akt phosphorylation, increased apoptotic markers (Bax protein and cleaved caspase-3) and augmented superoxide anion production. CONCLUSIONS: In addition to reduced insulin secretion, long-term pravastatin treatment induces insulin resistance and muscle wasting. These results suggest that the diabetogenic effect of statins is linked to the appearance of myotoxicity induced by oxidative stress, impaired insulin signalling, proteolysis and apoptosis.

Metformin treatment modulates the tumour-induced wasting effects in muscle protein metabolism minimising the cachexia in tumour-bearing rats.

BACKGROUND: Cancer-cachexia state frequently induces both fat and protein wasting, leading to death. In this way, the knowledge of the mechanism of drugs and their side effects can be a new feature to treat and to have success, contributing to a better life quality for these patients. Metformin is an oral drug used in type 2 diabetes mellitus, showing inhibitory effect on proliferation in some neoplastic cells. For this reason, we evaluated its modulatory effect on Walker-256 tumour evolution and also on protein metabolism in gastrocnemius muscle and body composition. METHODS: Wistar rats received or not tumour implant and metformin treatment and were distributed into four groups, as followed: control (C), Walker 256 tumour-bearing (W), metformin-treated (M) and tumour-bearing treated with metformin (WM). Animals were weighed three times a week, and after cachexia state has been detected, the rats were euthanised and muscle and tumour excised and analysed by biochemical and molecular assays. RESULTS: Tumour growth promoted some deleterious effects on chemical body composition, increasing water and decreasing fat percentage, and reducing lean body mass. In muscle tissue, tumour led to a decreased protein synthesis and an increased proteolysis, showing the higher activity of the ubiquitin-proteasome pathway. On the other hand, the metformin treatment likely minimised the tumour-induced wasting state; in this way, this treatment ameliorated chemical body composition, reduced the higher activities of proteolytic enzymes and decreased the protein waste. CONCLUSION: Metformin treatment not only decreases the tumour growth but also improves the protein metabolism in gastrocnemius muscle in tumour-bearing rats.

Leucine-rich diet improves the serum amino acid profile and body composition of fetuses from tumor-bearing pregnant mice.

Pregnancy is a complex process that can be jeopardized when associated with cancer, because of the coexistence of two complex metabolic conditions: a fetus and cancer. The aim of this study was to evaluate fetal growth in association with cancer development as well as the indirect effects produced by tumors in pregnant mice subjected to a leucine-rich diet, knowing that leucine supplementation can minimize the tumor effects by acting as a cell signaling agent to improve the protein synthesis process. We evaluated fetuses (n = 6) from NMRI pregnant mice fed either a control or a leucine-rich diet in either the presence or absence of an MAC16 colon adenocarcinoma or ascitic fluid inoculation. The fetal serum amino acids were separated using high-performance liquid chromatography, and fetal cytokine levels were analyzed using a microsphere-based multiplex immunoassay (Luminex xMAP). Fetal body composition was measured as the water, fat, and protein total content and total serum protein, albumin, and glucose content. Tumor growth resulted in a severe reduction in fetal body weight and protein content and increased fetal resorption, associated with placental weight decrease; these effects were minimized by a leucine-rich diet. Serum total protein and glucose content were reduced in fetuses from tumor-bearing dams but were reverted by nutritional supplementation. The serum amino acid profiles differed significantly between the tumor-bearing mice fed with a leucine-rich diet and controls. Certain tumor effects were reproduced in fetuses from ascitic fluid-injected dams, suggesting indirect effects of tumor growth. We conclude that certain effects of tumor growth can be mimicked by ascitic fluid injection and can be modulated by a leucine-rich diet.

Naringin inhibits tumor growth and reduces interleukin-6 and tumor necrosis factor α levels in rats with Walker 256 carcinosarcoma.

The flavonoid naringin is a polyphenolic compound that naturally occurs in citrus. Patients with cancer generally present features of malnutrition and cachexia. Levels of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) are raised in patients with cancer. This study was designed to analyze the in vivo effect of naringin in the therapeutic treatment of rats bearing Walker 256 carcinosarcoma (W256). Rats were treated intraperitoneally with different doses of naringin (10, 25 and 35 mg/kg), for 50 days. At 25 mg/kg, naringin inhibited tumor growth by ~75%. With this treatment, TNF-α and IL-6 levels decreased (p<0.05) in comparison with the control. In addition, two rats presented complete tumor regression. Inhibition of tumor growth, survival increase and the reduction of TNF-α and IL-6 levels in rats bearing W256 treated with naringin strongly suggest that this compound has potential as an anticarcinogenic drug.

Inhibition of tumor growth by quercetin with increase of survival and prevention of cachexia in Walker 256 tumor-bearing rats.

Quercetin, a flavonoid abundantly present in fruit, vegetables, wine and tea, has revealed several properties such as antioxidant, antiproliferative and anticancer. Cachexia is a poorly understood syndrome present in already compromised cancer patients, decreasing the quality of life and increasing mortality. Many studies have been performed in an attempt to discover an effective treatment for cachexia, but none of the tested therapies has fulfilled expectations. The objective of the present study was to analyze the effect of quercetin in the therapeutic treatment of cachexia and reversion of tumor growth in rats bearing Walker 256 carcinosarcoma (W256). Rats bearing W256 were treated daily with I.P. quercetin injections, at different doses (10, 15, 25 and 35 mg/kg). The results show that 10 mg/kg quercetin inhibited tumor growth by about 50% (ED(50)) when compared with controls (CTR). Moreover, two animals of this group presented complete tumor regression. Matrix metalloproteinase-2 (MMP-2) activity and vascular endothelial growth factor (VEGF) expression decreased in rats bearing W256 treated with 10 mg/kg quercetin when compared with CTR. Thus, the inhibition of tumor growth, survival increase, decrease of MMP-2 and VEGF levels and reduction of cachexia in animals treated with quercetin strongly support the anticancer function of this flavonoid.

Physical exercise and a leucine-rich diet modulate the muscle protein metabolism in Walker tumor-bearing rats.

Leucine-supplemented diet can recover lean body mass and preserve muscle protein mass. Additionally, physical exercise can be an excellent alternative to improve the rehabilitation of cancer patients. Knowing these facts, we examined the effects of a leucine-rich diet with or without physical aerobic exercise on muscle protein metabolism in Walker tumor-bearing rats. Young rats were divided into 4 groups that did or did not perform light aerobic exercise (swim training) and were on a leucine-rich diet or a control diet for 2 mo. After this time, these animals were implanted or not with tumors (subcutaneously) following groups for either control diet or leucine-rich diet fed rats: control, trained, tumor-bearing, and trained tumor-bearing. Twenty-one days after implantation, the tumor growth induced a decrease in the muscle protein synthesis and increased the catabolic process, which was associated with an increase in the expression of the ubiquitin-proteasome subunits (20S, 19S, and 11S). In contrast, the exercise program minimized the muscle degradation process and increased muscle myosin content. Additionally, leucine supplementation also modulated proteasome subunits, especially the 19S and 11S. In summary, the exercise has beneficial effects by reducing tumor growth, leading to an improvement in protein turnover especially when in conjunction with a leucine-rich diet.

Dietary fatty acid quality affects AR and PPARgamma levels and prostate growth.

BACKGROUND: Fatty acids are among the most important nutritional factors associated with the ethiopathogenesis of prostate cancer, therefore the main objective of this work was to evaluate the effect of quality of fatty acid on the rat ventral prostate growth, tissue organization, and expression of androgen receptor (AR) and peroxisome proliferation activator receptor gamma (PPARgamma). METHODS: Wistar rats were distributed into five groups, which were fed isocaloric normolipidic diets containing soybean oil (7% Control), linseed (7% or 3.5% linseed plus 3.5% soybean oil) and rendered pork fat (7% or 3.5% lard plus 3.5% soybean oil) for 10 weeks after weaning. RESULTS: At the end of treatment, the experiments demonstrated that lard and linseed oil caused opposite effects on prostatic growth. While the lard promoted an increase in prostatic weight associated to epithelial hyperplasia (confirmed by stereology); the linseed resulted in a significantly lighter organ. Immunohistochemistry and Western blotting demonstrated increased expression of AR and PPARgamma in groups fed with lard diet, while linseed oil promoted a decrease. CONCLUSIONS: Prostatic growth is influenced by dietary fatty acids with concurrent variation in the expression of AR and PPARgamma. PPARgamma might represent the link between diet and prostate growth and AR expression and function. Since the levels of testosterone were altered it is also possible that prostatic changes are secondary to systemic effects of the diet.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes.

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

Leucine-rich diet alters the eukaryotic translation initiation factors expression in skeletal muscle of tumour-bearing rats.

BACKGROUND: Cancer-cachexia induces a variety of metabolic disorders on protein turnorver, decreasing protein synthesis and increasing protein degradation. Conversely, insulin, other hormones, and branched-chain amino acids, especially leucine, stimulate protein synthesis and modulate the activity of translation initiation factors involved in protein synthesis. Since the tumour effects are more pronounced when associated with pregnancy, ehancing muscle-wasting proteolysis, in this study, the influence of a leucine-rich diet on the protein synthesis caused by cancer were investigated. METHODS: Pregnant rats with or without Walker 256 tumour were distributed into six groups. During 20 days of experiment, three groups were fed with a control diet: C--pregnant control, W--tumour-bearing, and P--pair-fed, which received the same amount of food as ingested by the W group; three other groups of pregnant rats were fed a leucine-rich diet: L--pregnant leucine, WL--tumour-bearing, and PL--pair-fed, which received the same amount of food as ingested by the WL group. RESULTS: The gastrocnemius muscle of WL rats showed increased incorporation of leucine in protein compared to W rats; the leucine-rich diet also prevented the decrease in plasma insulin normally seen in W. The expression of translation initiation factors increased when tumour-bearing rats fed leucine-rich diet, with increase of ~35% for eIF2alpha and eIF5, ~17% for eIF4E and 20% for eIF4G; the expression of protein kinase S6K1 and protein kinase C was also highly enhanced. CONCLUSION: The results suggest that a leucine-rich diet increased the protein synthesis in skeletal muscle in tumour-bearing rats possibly through the activation of eIF factors and/or the S6kinase pathway.

Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress.

Muscle wasting in cancer cachexia is associated with increased levels of malondialdehyde (MDA) in gastrocnemius muscles, suggesting an increased oxidative stress. To determine whether oxidative stress contributes to muscle protein catabolism, an in vitro model system, consisting of C2C12 myotubes, was treated with either 0.2 mM FeSO4, 0.1 mM H2O2, or both, to replicate the rise in MDA content in cachexia. All treatments caused an increased protein catabolism and a decreased myosin expression. There was an increase in the proteasome chymotrypsin-like enzyme activity, while immunoblotting showed an increased expression of the 20S proteasome alpha-subunits, p42, and the ubiquitin-conjugating enzyme, E214k. These results show that mild oxidative stress increases protein degradation in skeletal muscle by causing an increased expression of the major components of the ubiquitin-proteasome pathway.