Branched-Chain Amino Acids Enhance Premature Senescence through Mammalian Target of Rapamycin Complex I-Mediated Upregulation of p21 Protein

Branched-chain amino acids (BCAAs) have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR) links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer''s ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.     

Plasma amino acids in four models of experimental liver injury in rats

Summary We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.Abbreviations BCAA branched-chain amino acids (i.e. valine, leucine and isoleucine) - AAA aromatic amino acids (i.e. tyrosine and phenylalanine)     

Significance of branched chain amino acids as possible stimulators of hepatocyte growth factor

Amino acids can serve as regulatory molecules that modulate numerous cellular functions. Branched chain amino acids (BCAAs) are known to exert influences on cellular metabolism, amino acid transport, protein turn over, and gene expression. However, the mechanisms involved in the specific effect of BCAAs have not been clarified. BCAA supplementation therapy is a current treatment for patients with liver cirrhosis, therefore, specific BCAA activities should be examined. Hepatocyte growth factor (HGF) is considered to be a pleiotropic factor, and is reported to modulate gene expression and to stimulate the proliferation and functions of many cell types, including hepatocytes. A potential application of HGF for several types of diseases has been postulated. Here, we describe the potential of BCAAs as a therapeutic agent that acts through the induction of HGF production in the liver.     

Hepatic antioxidant enzymes and lipid peroxidation in carbon tetrachloride-induced liver cirrhosis in rats

Liver cirrhosis was induced in rats by the combined action of oral phenobarbitone and inhalations of carbon tetrachloride vapors. These rats manifested hepatosplenomegaly, hypoalbuminemia, and 2- to 17-fold elevations in serum transaminases and alkaline phosphatase levels. The hepatic antioxidant enzymes, superoxide dismutase and catalase, showed 28 and 60% decreases, respectively. There was, however, no increase in the hepatic lipid peroxidation. These studies suggest that in cirrhosis liver cell damage may result due to the direct attack of the oxygen free radicals. Lipid peroxidation in the liver may not be a prerequisite for the development of cirrhosis, as is generally believed.     

GC-MS Based Plasma Metabolomics for Identification of Candidate Biomarkers for Hepatocellular Carcinoma in Egyptian Cohort

This study evaluates changes in metabolite levels in hepatocellular carcinoma (HCC) cases vs. patients with liver cirrhosis by analysis of human blood plasma using gas chromatography coupled with mass spectrometry (GC-MS). Untargeted metabolomic analysis of plasma samples from participants recruited in Egypt was performed using two GC-MS platforms: a GC coupled to single quadruple mass spectrometer (GC-qMS) and a GC coupled to a time-of-flight mass spectrometer (GC-TOFMS). Analytes that showed statistically significant changes in ion intensities were selected using ANOVA models. These analytes and other candidates selected from related studies were further evaluated by targeted analysis in plasma samples from the same participants as in the untargeted metabolomic analysis. The targeted analysis was performed using the GC-qMS in selected ion monitoring (SIM) mode. The method confirmed significant changes in the levels of glutamic acid, citric acid, lactic acid, valine, isoleucine, leucine, alpha tocopherol, cholesterol, and sorbose in HCC cases vs. patients with liver cirrhosis. Specifically, our findings indicate up-regulation of metabolites involved in branched-chain amino acid (BCAA) metabolism. Although BCAAs are increasingly used as a treatment for cancer cachexia, others have shown that BCAA supplementation caused significant enhancement of tumor growth via activation of mTOR/AKT pathway, which is consistent with our results that BCAAs are up-regulated in HCC.     

Branched-Chain Amino Acid Supplementation Reduces Oxidative Stress and Prolongs Survival in Rats with Advanced Liver Cirrhosis

Long-term supplementation with branched-chain amino acids (BCAA) is associated with prolonged survival and decreased frequency of development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis. However, the pharmaceutical mechanism underlying this association is still unclear. We investigated whether continuous BCAA supplementation increases survival rate of rats exposed to a fibrogenic agent and influences the iron accumulation, oxidative stress, fibrosis, and gluconeogenesis in the liver. Further, the effects of BCAA on gluconeogenesis in cultured cells were also investigated. A significant improvement in cumulative survival was observed in BCAA-supplemented rats with advanced cirrhosis compared to untreated rats with cirrhosis (P<0.05). The prolonged survival due to BCAA supplementation was associated with reduction of iron contents, reactive oxygen species production and attenuated fibrosis in the liver. In addition, BCAA ameliorated glucose metabolism by forkhead box protein O1 pathway in the liver. BCAA prolongs survival in cirrhotic rats and this was likely the consequences of reduced iron accumulation, oxidative stress and fibrosis and improved glucose metabolism in the liver.     

Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver

After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.     

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver–skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.     

Branched‐chain amino acids prevent insulin‐induced hepatic tumor cell proliferation by inducing apoptosis through mTORC1 and mTORC2‐dependent mechanisms

Branched‐chain amino acids (BCAA) supplementation has been reported to suppress the incidence of liver cancer in obese patients with liver cirrhosis or in obese and diabetic model animals of carcinogenesis. Whether BCAA directly suppresses cell proliferation of hepatic tumor cells under hyperinsulinemic condition remain to be defined. The aim of this study was to investigate the effects of BCAA on insulin‐induced proliferation of hepatic tumor cells and determine the underlying mechanisms. BCAA suppressed insulin‐induced cell proliferation of H4IIE, HepG2 cells. In H4IIE cells, BCAA did not affect cell cycle progression but increased apoptosis by suppressing expressions of anti‐apoptotic genes and inducing pro‐apoptotic gene via inactivation of PI3K/Akt and NF‐κB signaling pathways. Further studies demonstrated that BCAA inhibited PI3K/Akt pathway not only by promoting negative feedback loop from mammalian target of rapamycin complex 1 (mTORC1)/S6K1 to PI3K/Akt pathway, but also by suppressing mTORC2 kinase activity toward Akt. Our findings suggest that BCAA supplementation may be useful to suppress liver cancer progression by inhibiting insulin‐induced PI3K/Akt and subsequent anti‐apoptotic pathway, indicating the importance of BCAA supplementation to the obese patients with advanced liver disease. J. Cell. Physiol. 227: 2097–2105, 2012. © 2011 Wiley Periodicals, Inc.     

Saikosaponin-d attenuates the development of liver fibrosis by preventing hepatocyte injury.

Treatment of liver fibrosis and cirrhosis remains a challenging field. Hepatocyte injury and the activation of hepatic stellate cells are the 2 major events in the development of liver fibrosis and cirrhosis. It is known that several Chinese herbs have significant beneficial effects on the liver; therefore, the purpose of the present study was to investigate the therapeutic effect of saikosaponin-d (SSd) on liver fibrosis and cirrhosis. A rat model of liver fibrosis was established using the dimethylnitrosamine method. Liver tissue and serum were used to examine the effect of SSd on liver fibrosis. A hepatocyte culture was also used to investigate how SSd can protect hepatocytes from oxidative injury induced by carbon tetrachloride. The results showed that SSd significantly reduced collagen I deposition in the liver and alanine aminotransferase level in the serum. Moreover, SSd decreased the content of TGF-beta1 in the liver, which was significantly elevated after dimethylnitrosamine induced liver fibrosis. Furthermore, SSd was able to alleviate hepatocyte injury from oxidative stress. In conclusion, SSd could postpone the development of liver fibrosis by attenuating hepatocyte injury.     

Oral Hepatoprotective Ability Evaluation of Purple Sweet Potato Anthocyanins on Acute and Chronic Chemical Liver Injuries

In recent years, chemical liver injury cases increased significantly in Asian countries, and the imbalance in redox system was believed to be the main cause. Purple sweet potato anthocyanins (PSPA) have been shown to exert antioxidant activity and oxidative-stress-associated functional protein modulation through various signaling pathways, so it is considered to have the potential of liver injury preventive activity. In order to evaluate the hepatoprotective potency of PSPA according to its free radical scavenging and antioxidant effects, three acute chemical liver injury models were set up with ethanol, acetaminophen and carbon tetrachloride. PSPA at moderate and high doses obviously attenuated the tested serum biomarker levels and liver index in our experiments. Besides, one chronic liver injury model set up with carbon tetrachloride was also applied, in which PSPA was orally administrated after the liver damage had been formed. Both the serum biomarker levels and histopathological analysis showed that PSPA was able to attenuated chronic liver injury. Our experimental results demonstrated the potential of PSPA as an oral hepatoprotective agent against chemical liver injury from food plant.     

Leucine and Protein Metabolism in Obese Zucker Rats

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-¹⁴C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

Branched-chain amino acid supplementation during bed rest: effect on recovery.

Bed rest is associated with a loss of protein from the weight-bearing muscle. The objectives of this study are to determine whether increasing dietary branched-chain amino acids (BCAAs) during bed rest improves the anabolic response after bed rest. The study consisted of a 1-day ambulatory period, 14 days of bed rest, and a 4-day recovery period. During bed rest, dietary intake was supplemented with either 30 mmol/day each of glycine, serine, and alanine (group 1) or with 30 mmol/day each of the three BCAAs (group 2). Whole body protein synthesis was determined with U-(15)N-labeled amino acids, muscle, and selected plasma protein synthesis with l-[(2)H(5)]phenylalanine. Total glucose production and gluconeogenesis from alanine were determined with l-[U-(13)C(3)]alanine and [6,6-(2)H(2)]glucose. During bed rest, nitrogen (N) retention was greater with BCAA feeding (56 +/- 6 vs. 26 +/- 12 mg N. kg(-1). day(-1), P < 0.05). There was no effect of BCAA supplementation on either whole body, muscle, or plasma protein synthesis or the rate of 3-MeH excretion. Muscle tissue free amino acid concentrations were increased during bed rest with BCAA (0.214 +/- 0.066 vs. 0.088 +/- 0.12 nmol/mg protein, P < 0.05). Total glucose production and gluconeogenesis from alanine were unchanged with bed rest but were significantly reduced (P < 0.05) with the BCAA group in the recovery phase. In conclusion, the improved N retention during bed rest is due, at least in part, to accretion of amino acids in the tissue free amino acid pools. The amount accreted is not enough to impact protein kinetics in the recovery phase but does improve N retention by providing additional essential amino acids in the early recovery phase.     

4-羟基苯并恶唑-2-酮对四氯化碳致小鼠急性肝损伤保护作用

日的研究4一羟基苯并恶唑-2-酮（4-hydroxy-2-benzoxazolone,HBOA）对四氯化碳所致小鼠急性肝损伤的保护作用,并探讨其疗效机制。方法采用腹腔注射四氯化碳（carbonte trachloride,cch）制备小鼠急性肝损伤模型,HBOA灌胃给药,检测小鼠血清中的乳酸脱氢酶（LDH）活性以及肝组织中过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）含量,并用免疫组化法观察肿瘤坏死因子（TNF-a）的表达情况。结果HBOA能明显降低CCh致急性肝损伤小鼠血清LDH活性,同时升高肝组织中CAT、GSH-Px的活性并降低肝组织中TNF-a的表达。结论HBOA对CCh所致小鼠急性肝损伤有一定的保护作用。     

Disordered branched chain amino acid catabolism in pancreatic islets is associated with postprandial hypersecretion of glucagon in diabetic mice

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes. We previously reported that postprandial hyperglucagonemia is more obvious than fasting hyperglucagonemia in type 2 diabetes patients. However, which nutrient stimulates glucagon secretion in the diabetic state and the underlying mechanism after nutrient intake are unclear. To answer these questions, we measured plasma glucagon levels in diabetic mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using islets isolated from diabetic mice and palmitate-treated islets. In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets. We found that protein, but not carbohydrate or lipid, increased plasma glucagon levels in diabetic mice. Among amino acids, BCAAs, but not the other essential or nonessential amino acids, increased plasma glucagon levels. BCAAs also directly increased the intracellular calcium concentration in α cells. When BCAAs transport was suppressed by an inhibitor of system L-amino acid transporters, glucagon secretion was reduced even in the presence of BCAAs. We also found that the expression levels of BCAA catabolism-related enzymes and their metabolite contents were altered in diabetic islets and palmitate-treated islets compared to control islets, indicating disordered BCAA catabolism in diabetic islets. Furthermore, BCKDK inhibitor BT2 suppressed BCAA-induced hypersecretion of glucagon in diabetic islets and palmitate-treated islets. Taken together, postprandial hypersecretion of glucagon in the diabetic state is attributable to disordered BCAA catabolism in pancreatic islet cells.     

Plasma amino acid imbalance in alcoholic liver cirrhosis

Plasma amino acid concentrations and plasma glucagon and serum insulin levels were studied in male patients with compensated alcoholic and nonalcoholic liver cirrhosis. Age, nutritional status, and liver function tests were similar in both groups; none of the patients presented hepatic encephalopathy. Plasma valine and leucine concentrations were lower, and tyrosine, higher in alcoholic than nonalcoholic liver cirrhosis. As a result, the molar ratios of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) were reduced markedly in this group. Although correlation coefficients comparing BCAA/AAA ratios and KICG in alcoholic and nonalcoholic liver cirrhosis were similar, a steeper regression line was observed in alcoholics. Plasma glucagon and proline levels were significantly higher in alcoholic than nonalcoholic liver cirrhosis, the former correlated with AAA concentrations only in alcoholic liver cirrhosis, but not with BCAA levels. These results indicated that alcoholic liver cirrhosis presented a more deranged plasma amino acid pattern than nonalcoholic, and the amino acid imbalances, except for depressed BCAA and elevated proline, were derived, in part, from the hyperglucagonemia.     

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF- production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF- and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhances TNF- expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.