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Dietary L-arginine supplementation reduces fat mass in diet-induced obese rats
Abstract
This study was conducted to test the hypothesis that dietary arginine supplementation reduces fat mass in diet-induced obese rats. Male Sprague-Dawley rats were fed either low- or high-fat diets for 15 wks (16 rats/diet). Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing either 1.51% L-arginine-HCl or 2.55% alanine (isonitrogenous control) (n=8/treatment group). Twelve weeks after the initiation of the arginine treatment, rats were euthanized to obtain tissues for biochemical analyses. Results were statistically analyzed as a 2x2 factorial experimental design using ANOVA. High-fat diet increased the mass of white adipose tissues at different anatomical locations by 49-96% compared to the low-fat diet. Concentrations of serum cholesterol as well as lipids in skeletal muscle and liver were higher in obese rats than in lean rats. L-Arginine supplementation reduced white adipose tissue mass by 20-40% while increasing brown adipose tissue mass by 15-20%. In addition, arginine treatment decreased adipocyte size, serum concentrations of glucose, triglycerides and leptin, improved glucose tolerance, and enhanced glucose and oleic acid oxidation in skeletal muscles. The mRNA levels for hepatic fatty acid synthase and stearoyl-CoA desaturase were reduced, but mRNA levels for hepatic AMP-activated protein kinase (AMPK), PPAR coactivator-1 and carnitine palmitoyltransferase I (CPT-I) as well as muscle CPT-I were increased in response to the arginine treatment. Subsequent experiments were conducted with cell models to define the direct effects of arginine on energy-substrate metabolism in insulin-sensitive cells. In BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes and 3T3-L1 mouse adipocytes, increasing extracellular concentrations of arginine from 0 to 400 µM increased AMPK expression as well as glucose and oleic acid oxidation. Inhibition of nitric oxide synthesis moderately attenuated the arginine-stimulated increases of substrate oxidation as well as AMPK and ACC phosphorylation in BNL CL.2 cells, but had no effect in C2C12 and 3T3-L1 cells. Collectively, these results suggest that arginine increases AMPK expression and energy-substrate oxidation in a cell-specific manner, thereby reducing fat mass in diet-induced obese rats. The findings have important implications for treating obesity in humans and companion animals as well as decreasing fat deposition in livestock species.
... Our previous studies using Zucker diabetic fatty rats (Fu et al. 2005) and diet-induced obese rats (Jobgen 2007;Jobgen et al. 2009) demonstrated that dietary supplementation with Arg increased glucose and fatty acid oxidation in skeletal muscle and white adipose tissue, therefore reducing body white-fat mass. Beneficial effects of Arg supplementation on reducing white fat, increasing lean tissue mass, and improving metabolic profiles have also been reported by many research groups for rodents and other members of the animal kingdom, including diet-induced obese rats (Alam et al. 2013;Miczke et al. 2015), mice (Sellmann et al. 2017), and sheep (Satterfield et al. 2012); growingfinishing pigs (Tan et al. 2011), poultry (Fouad et al. 2013), and fish (Li et al. 2020); individuals with diabetes mellitus (Mariotti 2020;Szlas et al. 2022); and obese persons (Boon et al. 2019;Khosroshahi et al. 2020;McNeal et al. 2018). ...
... Beneficial effects of Arg supplementation on reducing white fat, increasing lean tissue mass, and improving metabolic profiles have also been reported by many research groups for rodents and other members of the animal kingdom, including diet-induced obese rats (Alam et al. 2013;Miczke et al. 2015), mice (Sellmann et al. 2017), and sheep (Satterfield et al. 2012); growingfinishing pigs (Tan et al. 2011), poultry (Fouad et al. 2013), and fish (Li et al. 2020); individuals with diabetes mellitus (Mariotti 2020;Szlas et al. 2022); and obese persons (Boon et al. 2019;Khosroshahi et al. 2020;McNeal et al. 2018). Further, we found that the beneficial effect of Arg on metabolic health was mediated partially through the activation of hepatic AMP-activated protein kinase (AMPK) (Jobgen 2007;Jobgen and Wu 2022). However, the underlying biochemical mechanisms remain largely unknown (McKnight et al. 2010;Mariotti 2020;Szlas et al. 2022). ...
... 6.40 ± 1.12 b 6.88 ± 1.98 b 10.9 ± 1.38 b 25.7 ± 3.64 a 31.6 ± 3.05 a 0.0005 in the skeletal muscle and adipose tissue of obese rats (Fu et al. 2005;Jobgen 2007), as well whole-body glucose oxidation and insulin sensitivity in obese men (Boon et al. 2019). Because the BNL CL.2, C2C12, and 3T3-L1 cells were derived from the fetal mouse liver, the dystrophic adult mouse skeletal muscle, and the mouse embryonic fibroblast (Kuppusamy et al. 2021;Muller and Danner 2004), these cell lines may differ from the hepatocytes, skeletal muscle, Table 4 The effect of l-arginine on nitrite production in BNL CL.2, C2C12 and 3T3-L1 cells Cells were incubated for 48 h in customized DMEM containing 0, 15, 50, 100 or 400 µM of L-arginine. ...
Previous work has shown that dietary l-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400 µM) or 400 µM Arg + 0.5 mM NG-nitro-l-arginine methyl ester (L-NAME; an NOS inhibitor) for 48 h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P < 0.05) the oxidation of glucose and oleic acid to CO2 in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400 µM also stimulated (P < 0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P < 0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P < 0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P > 0.05) on these variables in C2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.
... Extensive studies in vitro have revealed that arginine stimulates lipolysis in adipocytes and promotes oxidation of long-chain fatty acids in insulin-sensitive tissues (1,8,(16)(17). For example, Jobgen (18) treated 3T3-L1 preadipocytes with different extracellular concentrations of L-arginine (50 -400 μM) and found that the rates of glucose and oleic acid oxidation were 45% and 40% greater, respectively, in the presence of arginine than in its absence. Similarly, in cultured human adipocytes, increasing extracellular concentrations of arginine from 0.4 to 2 mM increased the oxidation of 1 mM palmitate and 5 mM glucose by 32% and 51%, respectively (9). ...
... Interestingly, the reported effects of arginine on lipogenesis in white adipocytes have not been consistent. For example, in differentiated 3T3-L1 preadipocytes, the incorporation of oleic acid into lipids was increased by 190% in the presence of 400 μM arginine than in its absence (18), and high levels of arginine enhanced the expression of peroxisome proliferator-activated receptor (PPAR) gamma (a key regulator of adipogenesis) in preadipocytes and their differentiation (19). In contrast, arginine decreased the incorporation of palmitate and glucose into triglycerides in human adipocytes by 35% and 39%, respectively (9). ...
... Consistent with the reduction of white-fat mass, arginine treatment decreased circulating levels of glucose and nonesterified fatty acids and increased the oxidation of glucose and octanoate in abdominal and epididymal adipose tissues of obese rats (20). Moreover, arginine increased the expression of carnitine palmitoyltransferase 1 (CPT1), PCG-1alpha, and malonyl-CoA decarboxylase (MCD) in liver and depressed the expression of fatty acid synthase (FAS) and stearoyl coenzyme-A desaturase-1 (SCD1) (18). These results indicate a potent effect of arginine on inhibiting hepatic fatty acid synthesis from glucose. ...
As the nitrogenous precursor of nitric oxide, L-arginine regulates multiple metabolic pathways involved in the metabolism of fatty acids, glucose, amino acids, and proteins through cell signaling and gene expression. Specifically, arginine stimulates lipolysis and the expression of key genes responsible for activation of fatty acid oxidation to CO2 and water. The underlying mechanisms involve increases in the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), mitochondrial biogenesis, and the growth of brown adipose tissue growth. Furthermore, arginine regulates adipocyte-muscle crosstalk and energy partitioning via the secretion of cytokines and hormones. In addition, arginine enhances AMP-activated protein kinase (AMPK) expression and activity, thereby modulating lipid metabolism and energy balance toward the loss of triacylglycerols. Growing evidence shows that dietary supplementation with arginine effectively reduces white adipose tissue in Zucker diabetic fatty rats, diet-induced obese rats, growing-finishing pigs, and obese patients with type II diabetes. Thus, arginine can be used to prevent and treat adiposity and the associated metabolic syndrome.
... We found that dietary L-arginine (Arg) supplementation increased the oxidation of glucose and fatty acids in skeletal muscle, reduced body fat and serum concentrations of triglycerides, enhanced the mass of skeletal muscle, and improved insulin sensitivity in diet-induced obese (DIO) rats (Jobgen 2007;Jobgen et al. 2009). Consistent with our findings, dietary supplementation with 5% Arg for 8 weeks decreased abdominal fat and improved whole-body insulin sensitivity in adult rats fed a HF diet (Alam et al. 2013). ...
... Skeletal muscle also plays a crucial role in the homeostasis of glucose and fatty acids (Jobgen et al. 2006). Results from our research indicate that the rates of oxidation of these substrates in extensor digitorum longus muscle and soleus muscle, as measured with [U-14 C]glucose and [1-14 C]oleic acid, were decreased by high-fat feeding but increased in response to dietary Arg supplementation (Jobgen 2007). Arg treatment did not affect glucose or oleic acid oxidation in RP adipose tissue when expressed per 10 6 adipocytes (Jobgen 2007). ...
The goal of this study was to elucidate the molecular mechanisms responsible for the anti-obesity effect of L-arginine supplementation in diet-induced obese rats. Male Sprague–Dawley rats were fed either a low-fat or high-fat diet for 15 weeks. Thereafter, lean or obese rats were pair-fed their same respective diets and received drinking water containing either 1.51% L-arginine-HCl or 2.55% L-alanine (isonitrogenous control) for 12 weeks. Gene and protein expression of key enzymes in the metabolism of energy substrates were determined using real-time polymerase-chain reaction and western blotting techniques. The mRNA levels of hepatic fatty acid synthase and stearoyl-CoA desaturase were reduced (P < 0.05) but those of hepatic AMP-activated protein kinase-α (AMPKα), peroxisome proliferator activator receptor γ coactivator-1α, and carnitine palmitoyltransferase I (CPT-I), as well as skeletal muscle CPT-I were increased (P < 0.05) by L-arginine treatment. The protein expression and activity of hepatic AMPKα markedly increased (P < 0.05) but the activity of hepatic acetyl-CoA carboxylase (ACC) decreased (P < 0.05) in response to dietary L-arginine supplementation. Collectively, our results indicate that liver is the major target for the action of dietary L-arginine supplementation on reducing white-fat mass in diet-induced obese rats by inhibiting fatty acid synthesis and increasing fatty acid oxidation via the AMPK-ACC signaling pathway. Additionally, increased CPT-I expression in skeletal muscle may also contribute to the enhanced oxidation of long-chain fatty acids in L-arginine-supplemented rats.
... But in the liver, the infusion of Arg caused an increase in the expression of 3 of the 4 genes that encode for AMPK (PRKAA1, PRKAB1, and PRKAG1). The effect of Arg on AMPK has been studied previously, with inconsistent results including an increase in AMPK activity (at mRNA and protein level) in the liver, but not the adipose in diet-induced obese rats (Jobgen, 2007), no effect on AMPK in intestinal duodenal mucosal cells or longissimus muscle (Go et al., 2012), no effect on the activity of AMPKα1 in skeletal muscle (Linden et al., 2011), but a reduction in mRNA and protein levels of AMPK in the intestine of Hu sheep . The findings from our study and the studies described above suggest that the effect of Arg administration on AMPK are tissue-specific, which may be due to different sensitivity of specific tissues to Arg itself, or to the action of hormones such as insulin, NO, leptin, or ghrelin (Wu and Meininger, 2000;Kola et al., 2006;Tan et al., 2012). ...
... These findings, in different species, help to support the notion that Arg supplementation enhances lipolysis in the liver, by activating AMPK and the pathways that stimulate FA oxidation, resulting in a decrease in circulating FA. Similarly, in obese rats, dietary supplementation with Arg increased the mRNA expression and protein concentration of AMPK, together with the activity of CPT1 in the liver, which subsequently decreased the blood concentrations of glucose and triglycerides (Jobgen, 2007). ...
Arginine, one of the conditionally essential AA, has been reported to affect fat synthesis and metabolism in nonruminant animals by influencing adenosine monophosphate activated protein kinase (AMPK) in some organs. In dairy cows, the effect of Arg on milk fat production is not clear, and any potential mechanism that underlies the effect is unknown. We tested the hypothesis that Arg infusion would improve the production of milk fat, and explored possible mechanism that might underlie any effect. We used 6 healthy lactating cows at 20 ± 2 d in milk, in fourth parity, with a body weight of 508 ± 14 kg, body condition score of 3.0 ± 0, and a milk yield of 30.6 ± 1.8 kg/d (mean ± standard deviation). The cows were blocked by days in milk and milk yield and each cow received 3 treatments in a replicated 3 × 3 Latin square design, with each of the experimental periods lasting 7 d with a 14-d washout between each period. The treatments, delivered in random order, were (1) infusion of saline (control); (2) infusion of 0.216 mol/d of l-Arg in saline (Arg); (3) infusion of 0.868 mol/d of l-Ala in saline (the Arg and Ala treatments were iso-nitrogenous) through a jugular vein. On the last day of each experimental period, blood was sampled to measure insulin, nitric oxide, glucose, and nonesterified fatty acid, and the liver and mammary gland were biopsied to measure the expression of genes. Milk yield was recorded, and milk fat percentage was measured daily during each of the experimental periods. The yield and composition of fatty acid (FA) in milk was measured daily on the last 3 d during each of the experimental periods. The data were analyzed using a mixed model with treatment as a fixed factor, and cow, period, and block as random factors. The daily milk yield and milk fat yield when the cows were infused with Arg were 2.2 kg and 76 g, respectively, higher than that in control, and 1.8 kg and 111 g, respectively, higher than that in Ala. When the cows were infused with Arg they had higher concentration and yield of de novo synthesized FA, than when they received the control or Ala infusions, although milk fat percentage, daily feed intake, and the digestibility of nutrients were not affected by treatment. The serum concentration of nitric oxide and insulin were higher during Arg than during control or Ala, with no difference between control and Ala. In the liver, the expression of the genes coding for AMPK (PRKAA1, PRKAB1, and PRKAG1) and genes related to the oxidation of FA were higher during Arg than during control or Ala, whereas in the mammary gland the expression PRKAB1 was lowest, and the expression of genes involved in the synthesis of milk fat were highest, during Arg infusion. The results suggest the intravenous infusion of Arg enhanced the production of milk fat by promoting the de novo synthesis of FA and increasing milk yield.
... Insulin also promotes hepatic lipogenesis through several mechanisms and research has shown that arginine can affect lipid metabolism 10,13,45 . In our previous study, arginine supplementation (1.62% and 1.96%) increased whole body fat accretion in juvenile blunt snout bream 2 . ...
... In another study, arginine supplementation (7.20% of dietary protein) resulted in the highest fat gain in Atlantic salmon (Salmo salar) 45 , which supports our current experi- mental results. Our study and Andersen's study differ from studies carried out with mammals, which demonstrate a loss in fat mass after arginine supplementation [10][11][12][13] . To the best of our knowledge, most of the reports concern- ing the effect of arginine on fat gain in mammals have been based on observations of adult animals. ...
This study evaluated the mechanisms governing insulin resistance, glucose metabolism and lipogenesis in juvenile fish fed with graded levels of dietary arginine. The results showed that, compared with the control group (0.87%), 2.31% dietary arginine level resulted in the upregulation of the relative gene expression of IRS-1, PI3K and Akt in the insulin signaling pathway, while 2.70% dietary arginine level led to inhibition of these genes. 1.62% dietary arginine level upregulated glycolysis by increasing GK mRNA level; 2.70% dietary arginine level upregulated gluconeogenesis and resulted in high plasma glucose content by increasing PEPCK and G6P mRNA level. Furthermore, 2.70% dietary arginine level significantly lowered GLUT2 and increased PK mRNA levels. 1.62% dietary arginine level significantly upregulated ACC, FAS and G6PDH mRNA levels in the fat synthesis pathway and resulted in high plasma TG content. These results indicate that 1.62% dietary arginine level improves glycolysis and fatty acid synthesis in juvenile blunt snout bream. However, 2.70% dietary arginine level results in high plasma glucose, which could lead to negative feedback of insulin resistance, including inhibition of IRS-1 mRNA levels and activation of gluconeogenesis-related gene expression. This mechanism seems to be different from mammals at the molecular level.
... These pathways also involve activation of various proteins in the cytoplasm and the nucleus and have important implications for animal nutrition. For example, dietary supplementation with arginine increases expression of the AMPK gene in white adipose tissue of Zucker diabetic fatty rats (18) and diet-induced obese rats (39), as well as phosphorylation of the AMPK protein in skeletal muscle and liver of diet-induced obese rats (54). Furthermore, G-protein-coupled receptors participate in monosodium glutamate (MSG)-induced chemical sensing in the gastrointestinal tract, leading to increases in its motility, secretions, and absorptive ability (53). ...
Amino acids (AA) have enormous physiological importance, serving as building blocks for proteins and substrates for synthesis of low-molecular-weight substances. Based on growth or nitrogen balance, AA were traditionally classified as nutritionally essential or nonessential for animals. Although those AA that are not synthesized in eukaryotes (nutritionally essential AA, EAA) must be present in animal diets, nutritionally nonessential AA (NEAA) have long been ignored for all species. Emerging evidence shows that nonruminants cannot adequately synthesize NEAA or conditionally essential AA (CEAA) to realize their growth or anti-infection potential. Likewise, all preformed AA are needed for high-producing cows and rapidly growing ruminants. Many NEAA and CEAA (e.g., arginine, glutamine, glutamate, glycine, and proline) and certain EAA (e.g., leucine and tryptophan) participate in cell signaling, gene expression, and metabolic regulation. Thus, functions of AA beyond protein synthesis must be considered in dietary formulations to improve efficiency of nutrient use, growth, development, reproduction, lactation, and well-being in animals.
... It has been suggested that nitric oxide (NO), a signaling molecule produced from L-arginine by various isoforms of NO synthase (NOS), is involved in the regulation of hepatic gluconeogenesis. It was shown that physiological levels of NO stimulate glucose uptake and oxidation in insulin-sensitive tissues and inhibit the synthesis of glucose in target tissues (Jobgen, 2007). In this study increment of dietary arginine level enhanced T-NOS activity and this can be at least partially responsible for reduction of plasma glucose concentration at increased arginine levels. ...
A 9-week feeding trial was carried out to evaluate dietary arginine requirement of juvenile red sea bream. Six isonitrogenous and isocaloric diets (50% crude protein and 17.7 kJ g−1 gross energy) were formulated to contain graded levels of arginine including 1.42, 1.88, 2.22, 2.54, 3.08 and 3.43% of diet (2.84–6.86% of dietary protein), and fed triplicate groups of fish (13.3 ± 0.2 g) to apparent satiation twice daily. At the end of the feeding trial, fish fed ≥2.22% arginine showed significantly (P b 0.05) higher growth than those fed 1.42% arginine. Significant improvement in protein productive value was found at dietary arginine level of 2.54% compared to the fish fed 1.42% arginine. Significant reductions in whole-body and muscle lipid contents were found by increment of arginine level and whole-body protein increased significantly in fish fed 2.22–2.54% arginine compared to those fed 1.42% arginine. Plasma total protein level significantly was increased in fish fed 2.54–3.08% arginine, and alanine aminotransferase activity and glucose level were significantly decreased in fish fed 2.22–2.54% and ≥1.88% arginine, respectively, compared to the group fed 1.42% arginine. Significant improvements in lysozyme and myeloperoxidase activities and total immunoglobulin level were obtained by dietary arginine increment. Also, significantly higher total nitric oxide synthase activity was recorded at 3.08% arginine level in comparison to 1.42% arginine. A broken-line regression analysis on weight gain showed that the optimum dietary arginine level is 2.37% of diet (4.74% of dietary protein).
The aim of this study was to investigate the effects of different dietary levels of L-arginine on the growth performance, blood parameters, and lipogenic gene expression of Arian broiler chickens. For this purpose, 168 Arian broiler chicks (40.33±1.7 g) were assigned to four treatments with three replicates of14 birds each, according to completely randomized design. The experimental treatments consisted of 100, 124, 139, and 154% dietary arginine levels relative to the published requirements of Arian broilers. On 42 d of the experiment, blood samples were collected from two birds (six birds per treatment) for blood metabolite measurements. These birds were then euthanized for carcass evaluation and collection of tissue samples. Increasing dietary arginine levels reduced (p<0.05) the gene expression of fatty acid synthase, acetyl-coenzyme A carboxylase, and malic enzyme in the liver and lipoprotein lipase in the abdominal fat tissue, as well as abdominal fat relative weight. Increasing dietary arginine levels significantly increased (p<0.05) body weight, feed efficiency, carcass yield, breast and thigh relative weights, and glucose and HDL (high-density lipoprotein) blood levels, and reduced cholesterol, triglyceride, and LDL (low-density lipoprotein) blood levels. Since almost similar performance and carcass trait results were obtained both with 124 and 139% arginine levels, supplying Arian broiler diets with 124% arginine is suggested.
Keywords:
Arginine; Arian broiler chickens; Lipogenic gene expression; Performance
L-arginine is a nutritionally important amino acid that controls a wide spectrum of cellular functions and physiological processes, acting by itself or through its various metabolites. There are several factors that determine overall L-arginine homeostasis: dietary supplementation, endogenous de novo synthesis, whole-body protein turnover and its extensive metabolism. The destiny of L-arginine is determined by the complex network of enzymes and pathways differentially expressed according to health and disease status. Diabetes is characterized by reduced concentrations of L-arginine in plasma and many tissues, and failure of its metabolic effects. Emerging data suggest that oral supplementation of L-arginine exerts multiple beneficial effects on the complex etiological and pathophysiological basis of diabetes including: i) β-cell function and mass and ii) obesity and peripheral insulin resistance. This review emphasizes important aspects of L-arginine action which classifies this amino acid as a promising therapeutic approach in the treatment of diabetes.
l-Arginine (Arg) is synthesised from glutamine, glutamate, and proline via the intestinal-renal axis in humans and most other
mammals (including pigs, sheep and rats). Arg degradation occurs via multiple pathways that are initiated by arginase, nitric-oxide
synthase, Arg:glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline,
glutamate, creatine, and agmatine with each having enormous biological importance. Arg is also required for the detoxification
of ammonia, which is an extremely toxic substance for the central nervous system. There is compelling evidence that Arg regulates
interorgan metabolism of energy substrates and the function of multiple organs. The results of both experimental and clinical
studies indicate that Arg is a nutritionally essential amino acid (AA) for spermatogenesis, embryonic survival, fetal and
neonatal growth, as well as maintenance of vascular tone and hemodynamics. Moreover, a growing body of evidence clearly indicates
that dietary supplementation or intravenous administration of Arg is beneficial in improving reproductive, cardiovascular,
pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity,
and maintaining tissue integrity. Additionally, Arg or l-citrulline may provide novel and effective therapies for obesity, diabetes, and the metabolic syndrome. The effect of Arg
in treating many developmental and health problems is unique among AAs, and offers great promise for improved health and wellbeing
of humans and animals.
Obesity and type-II diabetes are growing major health issues worldwide. They are the leading risk factors for vascular insulin resistance, which plays an important role in the pathogenesis of cardiovascular disease, the leading cause of death in developed nations. Recent studies have shown that reduced synthesis of nitric oxide (NO; a major vasodilator) from L-arginine in endothelial cells is a major factor contributing to the impaired action of insulin in the vasculature of obese and diabetic subjects. The decreased NO generation results from a deficiency of (6R)-5,6,7,8-tetrahydrobiopterin [BH4; an essential cofactor for NO synthase (NOS)], as well as increased generation of glucosamine (an inhibitor of the pentose cycle for the production of NADPH, another cofactor for NOS) from glucose and L-glutamine. Accordingly, endothelial dysfunction can be prevented by (1) enhancement of BH4 synthesis through supplementation of its precursor (sepiapterin) via the salvage pathway; (2) transfer of the gene for GTP cyclohydrolase-I (the first and key regulatory enzyme for de novo synthesis of BH4); or (3) dietary supplementation of L-arginine (which stimulates GTP cyclohydrolase-I expression and inhibits hexosamine production). Modulation of the arginine-NO pathway by BH4 and arginine is beneficial for ameliorating vascular insulin resistance in obesity and diabetes.
Dietary L-arginine (Arg) supplementation reduces white-fat gain in diet-induced obese rats but the underlying mechanisms are unknown. This study tested the hypothesis that Arg treatment affects expression of genes related to lipid metabolism in adipose tissue. Four-week-old male Sprague-Dawley rats were fed a low-fat (LF) or high-fat (HF) diet for 15 weeks. Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing 1.51% Arg-HCl or 2.55% L: -alanine (isonitrogenous control). After 12 weeks of Arg supplementation, rats were euthanized to obtain retroperitoneal adipose tissue for analyzing global changes in gene expression by microarray. The results were confirmed by RT-PCR analysis. HF feeding decreased mRNA levels for lipogenic enzymes, AMP-activated protein kinase, glucose transporters, heme oxygenase 3, glutathione synthetase, superoxide dismutase 3, peroxiredoxin 5, glutathione peroxidase 3, and stress-induced protein, while increasing expression of carboxypeptidase-A, peroxisome proliferator activated receptor (PPAR)-alpha, caspase 2, caveolin 3, and diacylglycerol kinase. In contrast, Arg supplementation reduced mRNA levels for fatty acid binding protein 1, glycogenin, protein phosphates 1B, caspases 1 and 2, and hepatic lipase, but increased expression of PPARgamma, heme oxygenase 3, glutathione synthetase, insulin-like growth factor II, sphingosine-1-phosphate receptor, and stress-induced protein. Biochemical analysis revealed oxidative stress in white adipose tissue of HF-fed rats, which was prevented by Arg supplementation. Collectively, these results indicate that HF diet and Arg supplementation differentially regulate gene expression to affect energy-substrate oxidation, redox state, fat accretion, and adipocyte differentiation in adipose tissue. Our findings provide a molecular mechanism to explain a beneficial effect of Arg on ameliorating diet-induced obesity in mammals.
L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamine with or without 20 microM 6-diazo-5-oxo-L-norleucine (DON) or with 100 microM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20 microM) and azaserine (100 microM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca(2+) levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.
activity. However, expression studies need to be per- formed to confirm this. Taking into account the allelic frequencies of the genetic polymorphisms in CYP3A4 (10% heterozygous for CYP3A4*1B, 5.4% heterozygous for CYP3A4*2, and 2.2% heterozygous for CYP3A4*3), this implies that ;15% of the (Caucasian) population may carry a genetic polymorphism in this allele. Because genetic polymorphisms may exhibit strong differences in occurrence among different ethnic groups, other popula- tions need to be investigated to determine the allelic frequency of CYP3A4*3. In conclusion, we have described and validated a PCR-RFLP assay for the CYP3A4*3 allele. The frequency of this variant allele in the Caucasian population (1.1%) indicates that it might be important in predicting CYP3A4 activity based on genotype. Future research should be directed toward elucidating the effect of this polymor- phism on CYP3A4 enzymatic activity and toward estab- lishing whether this is solely a genetic, or also a func- tional, polymorphism.
It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes.
First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation.
SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation.
NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.
Arginine, an amino acid that is nutritionally essential for the fetus and neonate, is crucial for ammonia detoxification and the synthesis of molecules with enormous importance (including creatine, nitric oxide, and polyamines). A significant nutritional problem in preterm infants is a severe deficiency of arginine (hypoargininemia), which results in hyperammonemia, as well as cardiovascular, pulmonary, neurological, and intestinal dysfunction. Arginine deficiency may contribute to the high rate of infant morbidity and mortality associated with premature births. Although hypoargininemia in preterm infants has been recognized for more than 30 years, it continues to occur in neonatal intensive care units in the United States and worldwide. On the basis of recent findings, we propose that intestinal citrulline and arginine synthesis (the major endogenous source of arginine) is limited in preterm neonates owing to the limited expression of the genes for key enzymes (e.g., pyrroline-5-carboxylate synthase, argininosuccinate synthase and lyase), thereby contributing to hypoargininemia. Because premature births in humans occur before the normal perinatal surge of cortisol (an inducer of the expression of key arginine-synthetic enzymes), its administration may be a useful tool to advance the maturation of intestinal arginine synthesis in preterm neonates. Additional benefits of cortisol treatment may include the following: 1) allowing early introduction of enteral feeding to preterm infants, which is critical for intestinal synthesis of citrulline, arginine, and polyamines as well as for intestinal motility, integrity, and growth; and 2) shortening the expensive stay of preterm infants in hospitals as a result of accelerated organ maturation and the restoration of full enteral feeding. Further studies of fetal and neonatal arginine metabolism will continue to advance our understanding of the mechanisms responsible for the survival and growth of preterm infants. This new knowledge will be beneficial for designing the next generation of enteral and parenteral amino acid solutions to optimize nutrition and health in this compromised population.
There is increasing evidence that endogenous nitric oxide (NO) influences adipogenesis, lipolysis and insulin-stimulated glucose uptake. We investigated the effect of NO released from S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on basal and insulin-stimulated glucose uptake in adipocytes of normoglycaemic and streptozotocin (STZ)-induced diabetic rats. GSNO and SNAP at 0.2,0.5, and 1 mM brought about a concentration-dependent increase in basal and insulin-stimulated 2-deoxyglucose uptake in adipocytes of normoglycaemic and STZ-induced diabetic rats. SNAP at 1.0 mM significantly elevated basal 2-deoxyglucose uptake (115.8+/-10.4% compared with GSNO at the same concentration (116.1+/-9.4%; P less than 0.05) in STZ-induced diabetic rats. Conversely, SNAP at concentrations of 10 mM and 20 mM significantly decreased basal 2-deoxyglucose uptake by 50.0+/-4.5% and 61.5+/-7.2% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). GSNO at concentrations of 10 mM and 20 mM also significantly decreased basal 2-deoxyglucose uptake by 50.8+/-6.4% and 55.2+/-7.8% respectively in adipocytes of STZ-induced diabetic rats (P less than 0.05). These observations indicate that NO released from GSNO and SNAP at 1 mM or less stimulates basal and insulin-stimulated glucose uptake,and at concentrations of 10 mM and 20 mM inhibits basal glucose uptake. The additive effect of GSNO or SNAP, and insulin observed in this study could be due to different mechanisms and warrants further investigation.
The present study tested the hypothesis that nitric oxide (NO) is involved in the leptin-induced stimulation of lipolysis. The effect of intravenous (iv) administration of leptin (10, 100 and 1000 microg/kg body weight) or vehicle on serum NO concentrations and glycerol release from white adipocytes of Wistar rats was examined. One hour after injection, the three leptin doses tested increased serum NO concentrations 15.1%, 23.4% and 60.0%, respectively (P<.001 vs. baseline). The effect of leptin on NO concentrations was significantly dose dependent on linear trend testing (P=.0001). Simple linear regression analysis showed that the lipolytic rate measured was significantly correlated with serum NO concentrations (P=.0025; r=.52). In order to gain further insight into the potential underlying mechanisms, the effect of leptin on lipolysis was studied in the setting of nitric oxide synthase (NOS) inhibition or acute ganglionic blockade. The stimulatory effect of leptin on lipolysis was significantly decreased (P<.05) under NOS inhibition. On the contrary, the leptin-induced lipolysis was unaltered in pharmacologically induced ganglionic blockade. The lack of effect on isoproterenol-, forskolin- and dibutyryl-cyclic AMP-stimulated lipolysis suggests that leptin does not interfere with the signal transduction pathway at the beta-adrenergic receptor, the adenylate cyclase and the protein kinase A levels. These findings suggest that NO is a potential regulator of leptin-induced lipolysis.
Obesity is a multifactorial, chronic disorder that has reached epidemic proportions in most industrialized countries and is threatening to become a global epidemic. Obese patients are at higher risk from coronary artery disease, hypertension, hyperlipidemia, diabetes mellitus, cancers, cerebrovascular accidents, osteoarthritis, restrictive pulmonary disease, and sleep apnoea. In particular, visceral fat accumulation is usually accompanied by insulin resistance or type 2 diabetes mellitus, hypertension, hypertriglyceridemia, high uremic acid levels, low high density lipoprotein (HDL) cholesterol to define a variously named syndrome or metabolic syndrome. Metabolic syndrome is now considered a major cardiovascular risk factor in a large percentage of population in worldwide. Both obesity and metabolic syndrome are particularly challenging clinical conditions to treat because of their complex pathophysiological basis. Indeed, body weight represents the integration of many biological and environmental components and relationships among fat and glucose tolerance or blood pressure are not completely understood. Efforts to develop innovative anti-obesity drugs, with benefits for metabolic syndrome, have been recently intensified. In general two distinct strategies can be adopted: first, to reduce energy intake; second, to increase energy expenditure. Here we review some among the most promising avenues in these two fields of drug therapy of obesity and, consequently, of metabolic syndrome.
The First Law of Thermodynamics provides a framework for understanding the imbalance between energy intake and expenditure that produces obesity, but it does not help understand the role of genetics, the regulation of food intake, the distribution of body fat, the mechanisms by which diets work or the mechanism by which portion control has gotten out of control. In animals, increasing dietary fat increases body fat, and it is unlikely that humans escape this important biological rule. In epidemiological studies, increasing dietary fat is associated with increased prevalence of obesity probably by increasing the intake of energy dense foods. In the National Weight Loss Registry, three things were associated with weight loss: continued monitoring of food intake, lowering dietary fat intake, and increased exercise. The relation of dietary fat is most evident when physical activity is low. The speed of adaptation to dietary fat is increased by exercise. When dietary fat is reduced, weight is lost, but weight loss eventually plateaus. The rate of weight loss during the initial phase is about 1.6 g/day for each 1% decrease in fat intake. When dietary fat is replaced with olestra to reduce fat intake from 33% to 25% in obese men, weight loss continues for about 9 months reaching a maximum of nearly 6% of body weight and a loss of 18% of initial body fat. In the control group with a 25% reduced-fat diet, weight loss stopped after 3 months and was regained over the next 6 months, indicating the difficulty of adhering to a conventional low-fat diet. Thus, dietary fat is an important contributor to obesity in some people.
We investigated whether a dietary intake of arginine is associated with risk for cardiovascular disease as determined by levels of C-reactive protein (CRP).
We analyzed the Third National Health Nutrition and Examination Survey, a national public-use dataset collected between 1988 and 1994. Arginine intake was calculated from the 24-h dietary recall using the nutrient composition database of the University of Minnesota Nutrition Coordinating Center. A logistic regression model was used to evaluate the relation between arginine intake and serum levels of CRP while controlling for age, sex, race, exercise, total caloric intake, body mass index, smoking status, diabetes, hypertension, and fiber intake.
In the unadjusted model, the likelihoods of having a high level of CRP (>3.0 mg/L), from the lowest to the highest level of arginine intake, were 34.8%, 31.0%, 27.7%, and 18.4% respectively. Arginine intake below the median range was associated with higher levels of CRP (P < 0.05), and arginine intake above the median range was associated with lower levels of CRP (P < 0.05). In the adjusted regression, subjects in the highest level (90th percentile) of arginine intake were 30% less likely to have a CRP above 3.0 mg/L than were subjects with a median arginine intake (odds ratio= 0.70, 95% confidence interval = 0.56 to 0.88).
The results of this study show a relation between arginine intake and CRP level that persisted after controlling for factors associated with CRP. Individuals may be able to lower their risk for cardiovascular disease by consuming more arginine-rich foods such as nuts and fish.