Robin P. da Silva

Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

Novel insights on interactions between folate and lipid metabolism

Folate is an essential B vitamin required for the maintenance of AdoMet‐dependent methylation. The liver is responsible for many methylation reactions that are used for post‐translational modification of proteins, methylation of DNA, and the synthesis of hormones, creatine, carnitine, and phosphatidylcholine. Conditions where methylation capacity is compromised, including folate deficiency, are associated with impaired phosphatidylcholine synthesis resulting in non‐alcoholic fatty liver disease and steatohepatitis. In addition, folate intake and folate status have been associated with changes in the expression of genes involved in lipid metabolism, obesity, and metabolic syndrome. In this review, we provide insight on the relationship between folate and lipid metabolism, and an outlook for the future of lipid‐related folate research.

Creatine Supplementation Prevents the Accumulation of Fat in the Livers of Rats Fed a High-Fat Diet

The aim of the present study was to examine the effects of creatine supplementation on liver fat accumulation induced by a high-fat diet in rats. Rats were fed 1 of 3 different diets for 3 wk: a control liquid diet (C), a high-fat liquid diet (HF), or a high-fat liquid diet supplemented with creatine (HFC). The C and HF diets contained, respectively, 35 and 71% of energy derived from fat. Creatine supplementation involved the addition of 1% (wt:v) of creatine monohydrate to the liquid diet. The HF diet increased total liver fat concentration, liver TG, and liver TBARS and decreased the hepatic S-adenosylmethionine (SAM) concentration. Creatine supplementation normalized all of these perturbations. Creatine supplementation significantly decreased the renal activity of l-arginine:glycine amidinotransferase and plasma guanidinoacetate and prevented the decrease in hepatic SAM concentration in rats fed the HF diet. However, there was no change in either the phosphatidylcholine:phosphatidylethanolamine (PE) ratio or PE N-methyltransferase activity. The HF diet decreased mRNA for PPARα as well as 2 of its targets, carnitine palmitoyltransferase and long-chain acylCoA dehydrogenase. Creatine supplementation normalized these mRNA levels. In conclusion, creatine supplementation prevented the fatty liver induced by feeding rats a HF diet, probably by normalization of the expression of key genes of β-oxidation.

Amino acids and the regulation of methyl balance in humans.

Purpose of reviewTo outline recent advances in our understanding of the metabolic basis for the maintenance of cellular S-adenosylmethionine levels and, thus, for facilitating the many crucial methylation reactions in the body. Amino acids are intimately involved in these processes. Recent findingsThe application of stable-isotope methodology has permitted accurate estimation of the total transmethylation flux in humans. Chemical balance studies have identified the quantitatively major transmethylation reactions. New evidence points to a key role for deranged S-adenosylmethionine metabolism in the pathogenesis of liver disease. Mutations in key enzymes point to the importance of methyl metabolism in closure of the neural tube, synthesis of creatine and metabolic clearance of methionine. Dietary interventions designed to affect S-adenosylmethionine availability to pregnant mice have been shown to modulate the epigenetic DNA methylation of specific genes. SummaryThese findings are of relevance to the pathogenesis of neural tube defects as well as the interaction between a genetic polymorphism and nutritional status. They also address the issue of methyl group availability and epigenetic regulation. Finally, they are also relevant to the etiology of cirrhosis and steatohepatitis.

The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice

Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) catalyzes the synthesis of phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected against diet-induced obesity and insulin resistance. We investigated the role of PEMT in hepatic carbohydrate metabolism in chow-fed mice. A pyruvate tolerance test revealed that PEMT deficiency greatly attenuated gluconeogenesis. The reduction in glucose production was specific for pyruvate; glucose production from glycerol was unaffected. Mitochondrial PC levels were lower and PE levels were higher in livers from Pemt−/− compared with Pemt+/+ mice, resulting in a 33% reduction of the PC-to-PE ratio. Mitochondria from Pemt−/− mice were also smaller and more elongated. Activities of cytochrome c oxidase and succinate reductase were increased in mitochondria of Pemt−/− mice. Accordingly, ATP levels in hepatocytes from Pemt−/− mice were double that in Pemt+/+ hepatocytes. We observed a strong correlation between mitochondrial PC-to-PE ratio and cellular ATP levels in hepatoma cells that expressed various amounts of PEMT. Moreover, mitochondrial respiration was increased in cells lacking PEMT. In the absence of PEMT, changes in mitochondrial phospholipids caused a shift of pyruvate toward decarboxylation and energy production away from the carboxylation pathway that leads to glucose production.

Choline Supplementation Protects against Liver Damage by Normalizing Cholesterol Metabolism in Pemt/Ldlr Knockout Mice Fed a High-Fat Diet

Dietary choline is required for proper structure and dynamics of cell membranes, lipoprotein synthesis, and methyl-group metabolism. In mammals, choline is synthesized via phosphatidylethanolamine N-methyltransferase (Pemt), which converts phosphatidylethanolamine to phosphatidylcholine. Pemt(-/-) mice have impaired VLDL secretion and developed fatty liver when fed a high-fat (HF) diet. Because of the reduction in plasma lipids, Pemt(-/-)/low-density lipoprotein receptor knockout (Ldlr(-/-)) mice are protected from atherosclerosis. The goal of this study was to investigate the importance of dietary choline in the metabolic phenotype of Pemt(-/-)/Ldlr(-/-) male mice. At 10-12 wk of age, Pemt(+/+)/Ldlr(-/-) (HF(+/+)) and half of the Pemt(-/-)/Ldlr(-/-) (HF(-/-)) mice were fed an HF diet with normal (1.3 g/kg) choline. The remaining Pemt(-/-)/Ldlr(-/-) mice were fed an HF diet supplemented (5 g/kg) with choline (HFCS(-/-) mice). The HF diet contained 60% of calories from fat and 1% cholesterol, and the mice were fed for 16 d. HF(-/-) mice lost weight and developed hepatomegaly, steatohepatitis, and liver damage. Hepatic concentrations of free cholesterol, cholesterol-esters, and triglyceride (TG) were elevated by 30%, 1.1-fold and 3.1-fold, respectively, in HF(-/-) compared with HF(+/+) mice. Choline supplementation normalized hepatic cholesterol, but not TG, and dramatically improved liver function. The expression of genes involved in cholesterol transport and esterification increased by 50% to 5.6-fold in HF(-/-) mice when compared with HF(+/+) mice. Markers of macrophages, oxidative stress, and fibrosis were elevated in the HF(-/-) mice. Choline supplementation normalized the expression of these genes. In conclusion, HF(-/-) mice develop liver failure associated with altered cholesterol metabolism when fed an HF/normal choline diet. Choline supplementation normalized cholesterol metabolism, which was sufficient to prevent nonalcoholic steatohepatitis development and improve liver function. Our data suggest that choline can promote liver health by maintaining cholesterol homeostasis.

Creatine reduces hepatic TG accumulation in hepatocytes by stimulating fatty acid oxidation

Non-alcoholic fatty liver disease encompasses a wide spectrum of liver damage including steatosis, non-alcoholic steatohepatitis, fibrosis and cirrhosis. We have previously reported that creatine supplementation prevents hepatic steatosis and lipid peroxidation in rats fed a high-fat diet. In this study, we employed oleate-treated McArdle RH-7777 rat hepatoma cells to investigate the role of creatine in regulating hepatic lipid metabolism. Creatine, but not structural analogs, reduced cellular TG accumulation in a dose-dependent manner. Incubating cells with the pan-lipase inhibitor diethyl p-nitrophenylphosphate (E600) did not diminish the effect of creatine, demonstrating that the TG reduction brought about by creatine does not depend on lipolysis. Radiolabeled tracer experiments indicate that creatine increases fatty acid oxidation and TG secretion. In line with increased fatty acid oxidation, mRNA analysis revealed that creatine-treated cells had increased expression of PPARα and several of its transcriptional targets. Taken together, this study provides direct evidence that creatine reduces lipid accumulation in hepatocytes by the stimulation of fatty acid oxidation and TG secretion.

Synthesis of guanidinoacetate and creatine from amino acids by rat pancreas

Creatine is an important molecule involved in cellular energy metabolism. Creatine is spontaneously converted to creatinine at a rate of 1·7% per d; creatinine is lost in the urine. Creatine can be obtained from the diet or synthesised from endogenous amino acids via the enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). The liver has high GAMT activity and the kidney has high AGAT activity. Although the pancreas has both AGAT and GAMT activities, its possible role in creatine synthesis has not been characterised. In the present study, we examined the enzymes involved in creatine synthesis in the pancreas as well as the synthesis of guanidinoacetate (GAA) and creatine by isolated pancreatic acini. We found significant AGAT activity and somewhat lower GAMT activity in the pancreas and that pancreatic acini had measurable activities of both AGAT and GAMT and the capacity to synthesise GAA and creatine from amino acids. Creatine supplementation led to a decrease in AGAT activity in the pancreas, though it did not affect its mRNA or protein abundance. This was in contrast with the reduction of AGAT activity and mRNA and protein abundance in the kidney, suggesting that the regulatory mechanisms that control the expression of this enzyme in the pancreas are different from those in the kidney. Dietary creatine increased the concentrations of GAA, creatine and phosphocreatine in the pancreas. Unexpectedly, creatine supplementation decreased the concentrations of S-adenosylmethionine, while those of S-adenosylhomocysteine were not altered significantly.

Choline deficiency impairs intestinal lipid metabolism in the lactating rat.

Choline is a precursor to phosphatidylcholine (PC), a structural molecule in cellular membranes that is crucial for cell growth and function. PC is also required for the secretion of lipoprotein particles from liver and intestine. Choline requirements are increased during lactation when maternal choline is supplied to the offspring through breast milk. To investigate the effect of dietary choline on intestinal lipid metabolism during lactation, choline-supplemented (CS), phosphatidylcholine-supplemented (PCS) or choline-deficient (CD) diets were fed to dams during the suckling period. CD dams had lower plasma triacylglycerol, cholesterol and apoB in the fasted state and following a fat-challenge (P < .05). There was a higher content of neutral lipids and lower content of PC in the intestine of CD dams, compared with CS and PCS fed animals (P < .05). In addition, there was lower (P < .05) villus height in CD dams, which indicated a reduced absorptive surface area in the intestine. Choline is critical for the absorption of fat in lactating rats and choline deficiency alters intestinal morphology and impairs chylomicron secretion by limiting the supply of PC.

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague-Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.