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Biochemical and Molecular Actions of Nutrients

Dietary L-Arginine Supplementation Reduces Fat Mass in Zucker Diabetic Fatty Rats¹

Wenjiang J. Fu^*,, Tony E. Haynes^*, Ripla Kohli^*, Jianbo Hu^**, Wenjuan Shi^*, Thomas E. Spencer^**, Raymond J. Carroll^*,, Cynthia J. Meininger and Guoyao Wu^*,**,,2

^* Faculty of Nutrition, Department of Statistics, and ^** Department of Animal Science, Texas A&M University and Department of Medical Physiology, The Texas A&M University System Health Science Center, College Station, TX 77843

²To whom correspondence should be addressed. E-mail: g-wu{at}tamu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study was conducted to test the hypothesis that dietary supplementation of arginine, the physiologic precursor of nitric oxide (NO), reduces fat mass in the Zucker diabetic fatty (ZDF) rat, a genetically obese animal model of type-II diabetes mellitus. Male ZDF rats, 9 wk old, were pair-fed Purina 5008 diet and received drinking water containing arginine-HCl (1.51%) or alanine (2.55%, isonitrogenous control) for 10 wk. Serum concentrations of arginine and NO_x (oxidation products of NO) were 261 and 70% higher, respectively, in arginine-supplemented rats than in control rats. The body weights of arginine-treated rats were 6, 10, and 16% lower at wk 4, 7, and 10 after the treatment initiation, respectively, compared with control rats. Arginine supplementation reduced the weight of abdominal (retroperitoneal) and epididymal adipose tissues (45 and 25%, respectively) as well as serum concentrations of glucose (25%), triglycerides (23%), FFA (27%), homocysteine (26%), dimethylarginines (18–21%), and leptin (32%). The arginine treatment enhanced NO production (71–85%), lipolysis (22–24%), and the oxidation of glucose (34–36%) and octanoate (40–43%) in abdominal and epididymal adipose tissues. Results of the microarray analysis indicated that arginine supplementation increased adipose tissue expression of key genes responsible for fatty acid and glucose oxidation: NO synthase-1 (145%), heme oxygenase-3 (789%), AMP-activated protein kinase (123%), and peroxisome proliferator-activated receptor coactivator-1 (500%). The induction of these genes was verified by real-time RT-PCR analysis. In sum, arginine treatment may provide a potentially novel and useful means to enhance NO synthesis and reduce fat mass in obese subjects with type-II diabetes mellitus.

KEY WORDS: • arginine • nitric oxide • obesity • diabetes • gene expression

Obesity, which is a public health problem worldwide (1–3), is a major risk factor for insulin resistance, type II diabetes, atherosclerosis, stroke, hypertension, and some types of cancer (including colon and breast cancers) (4). Consequently, obesity claims an increasing number of lives and contributes to the tremendous cost of health care worldwide. In the United States alone, 300,000 people die of obesity-related diseases every year, the incidence of type II diabetes among children has increased 10-fold over the past decade, and obesity accounts for 6–8% of all health care expenditures (4). Unfortunately, clinicians have few tools with which to fight the obesity epidemic because current antiobesity drugs are not highly effective and are fraught with side effects (4,5). Thus, identifying new therapeutic interventions to reduce body fat mass will be extremely beneficial for human health.

Nitric oxide (NO), produced from L-arginine by NO synthase in virtually all mammalian cells (6), plays an important role in fat metabolism (7). Recent studies demonstrated that through an increase in the expression of peroxisome proliferator-activated receptor coactivator-1 (PGC-1),³ a master regulator of oxidative phosphorylation and mitochondrial biogenesis (8,9), endogenous NO triggers mitochondrial biogenesis in diverse cell types (10). Thus, NO may stimulate the oxidation of energy substrates (including fatty acids and glucose) in adipocytes, liver, skeletal muscle, heart, and the whole body. Consistent with this view is the finding that mice with the knock-out of endothelial NO synthase had higher body fat weight than wild-type mice with a normal expression of the protein although food intake did not differ between the 2 groups of mice (10). Similarly, an inhibition of systemic NO synthesis increased serum levels of triglycerides and body fat mass in rats (11). Although exogenous NO donors may either inhibit or increase lipolysis in incubated adipocytes depending on doses and experimental conditions (12,13), there is strong evidence that endogenous NO stimulates lipolysis in white adipose tissue, fatty acid oxidation in hepatocytes, and glucose utilization in skeletal muscle (14–17). On the basis of these findings, we hypothesized that dietary supplementation of arginine reduces fat accretion in the body. This hypothesis was tested using the Zucker diabetic fatty (ZDF) rat (a genetically obese animal model of type-II diabetes mellitus), which has a mutation in its leptin receptor (18).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Chemicals. Hexokinase, glucose-6-phosphate dehydrogenase, and nitrate reductase were purchased from Roche. HPLC-grade water and methanol were procured from Fisher Scientific. D-[U-¹⁴C]glucose and [1-¹⁴C]octanoate were purchased from American Radiolabeled Chemicals. Unless indicated, all other chemicals were obtained from Sigma Chemical.

    Rats. Male Zucker diabetic fatty (ZDF) rats (8 wk old) were obtained from Charles River and fed a Purina 5008 diet throughout the study. This diet contained 23.5% crude protein, 6.0% fat, 34.9% starch, 2.6% sucrose, 0.5% glucose plus fructose, 6.8% minerals, 3.8% fiber, and 17,364 kJ gross energy/kg. The contents of alanine, arginine, and lysine in the diet were 1.39, 1.44, and 1.40%, respectively. Rats were housed in a temperature- and humidity-controlled facility on a 12-h light:dark cycle. This study was approved by the Texas A&M University Animal Use and Care Committee.

At 9 wk of age, rats were assigned randomly to receive drinking water (distilled and deionized H₂O) containing either 1.51% L-arginine-HCl (equivalent to 1.25% arginine) or 2.55% L-alanine (isonitrogenous control) (n = 6 per treatment). This dose of arginine was chosen because previous studies showed that oral administration of arginine (1.25% in drinking water) prevented endothelial dysfunction in diabetic BB rats and streptozotocin-induced diabetic rats and had no adverse effect on nondiabetic rats (19,20). The free arginine base was not used because 1.25% arginine in distilled and deionized water (pH 6.5) yields an alkaline solution (pH 10.8), whereas 1.51% arginine-HCl had no effect on the pH of the solution. Arginine-supplemented rats were individually pair-fed a Purina 5008 diet on the basis of feed intake by alanine-treated rats (per kg body weight) to ensure similar intakes of energy and nutrients from the enteral diet. In a separate study, we found that when allowed free access to food, feed intake did not differ between alanine- and arginine-supplemented ZDF rats (T. E. Haynes, W. Shi, and G. Wu, unpublished data). Body weights and food intakes of rats were measured daily. Tail venous blood samples (0.2 mL) were obtained at 1600 h (10 h after the last feeding) from unanesthetized rats at wk 3, 6, and 10 after the initiation of arginine supplementation; serum was analyzed for amino acids, glucose, NO_x (nitrite and nitrate), and lipids. At the end of a 10-wk period of arginine supplementation, cardiac blood samples (8 mL) and tissues were obtained from rats for analysis of serum hormones and metabolic studies, respectively.

    Measurements of serum glucose, amino acids, NO_x, lipids, and hormones. Serum glucose was determined enzymatically using a fluorometric method involving hexokinase and glucose-6-phosphate dehydrogenase (21). Serum amino acids (including methylarginines and homocysteine) and NO_x (nitrite and nitrate) were measured using fluorometric HPLC methods, as described (20–22). Serum triglycerides and total cholesterol were determined enzymatically using assay kits #2780–250 and #2350–250, respectively, from ThermoDMA. Serum FFA were measured enzymatically using assay kit #994–75409 from Wako Chemicals. Serum insulin and growth hormone were analyzed using rat RIA kits #RI-13K and #RGH-45HK (Linco Research), respectively. Serum leptin, adiponectin, and total ghrelin were determined using human, mouse, and human RIA kits #HL-81HK, #MADP-60HK, and #GHRT-89HK (Linco), respectively.

    Determination of NO synthesis in freshly isolated adipose tissues. Abdominal (retroperitoneal) and epididymal adipose tissues (0.2 g) were rinsed with oxygenated (95% O₂/5% CO₂) Basal Medium Eagle (pH 7.4), and then incubated at 37°C for 6 h in 2 mL of freshly oxygenated (95% O₂/5% CO₂) Basal Medium Eagle (pH 7.4) containing 0.2 mmol/L L-arginine, 0.5 mmol/L L-glutamine, 20 mmol/L D-glucose, 100 kIU/L penicillin, 100 mg/L streptomycin, and 0.25 mg/L amphotericin B (20). At the end of a 6-h incubation period, media were analyzed for nitrite and nitrate (2 major stable end products of NO oxidation) by an HPLC method, as described (22). In all experiments, media incubated without cells were included as blanks.

    Determination of glucose and octanoate oxidation as well as glycerol release. Abdominal and epididymal adipose tissues (0.2 g) were rinsed with oxygenated (95% O₂/5% CO₂) Krebs bicarbonate buffer (pH 7.4) (23) and then incubated at 37°C for 2 h in 2 mL of oxygenated (95% O₂/5% CO₂) Krebs bicarbonate buffer (pH 7.4) containing 250 µg/L insulin, 20 mmol/L D-glucose, 2 mmol/L octanoate, and either D-[U-¹⁴C]glucose (0.5 µCi) or [1-¹⁴C]octanoate (0.5 µCi). Octanoate was chosen to determine fatty acid oxidation, because it does not require carnitine acyltransferases for entry into mitochondria (24). These concentrations of glucose and octanoate were chosen to mimic plasma levels of glucose (18 to 25 mmol/L) and FFA (1.5–2 mmol/L) in ZDF rats (18). In all experiments, media incubated without tissues were included as blanks. At the end of a 2-h incubation period, 0.2 mL of 1.5 mol/L HClO₄ was added through a rubber stopper into the medium, and ¹⁴CO₂ was collected in 0.2 mL NCS-II (Amersham) for analysis of radioactivity, as described (23). The specific activities of D-[U-¹⁴C]glucose and [1-¹⁴C]octanoate in the incubation medium were used to calculate the rate of ¹⁴CO₂ production. Neutralized medium was analyzed for glycerol enzymatically using a fluorometric method (25).

    Microarray analysis of gene expression. Frozen abdominal adipose tissue (2 g) was minced on dry ice and thawed in 5 mL TRIzol reagent (Life Technologies), and total RNA was isolated according to manufacturer’s recommendations. The quantity and quality of total RNA were determined using Agilent Technologies 2100 Bioanalyzer and by denaturing agarose gel electrophoresis (26). Subsequent experimental procedures were performed at the Genomics and Bioinformatics Core Facility in the NIEHS Center for Environmental and Rural Health of the Texas A&M University with use of the CodeLink^TM UniSet containing 35K rat gene probes (Amersham Biosciences). cRNA was synthesized according to manufacturer’s instructions using 10 µg of total RNA (26). The resultant double-stranded cDNA was purified on a QIAquick column (Qiagen), and cRNA was generated via an in vitro transcription reaction using T7 RNA polymerase and biotin-11-UTP (Perkin Elmer). cRNA was purified on an RNeasy column (Qiagen), quantified by spectrometry, and 10 µg was then fragmented by heating at 94°C for 20 min in the presence of magnesium. The fragmented cRNA was hybridized overnight at 37°C in hybridization buffer to a UniSet Rat Bioarray in an Innova 4080 shaking incubator (New Brunswick) at 300 rpm. After hybridization, arrays were washed in 0.75X Tris buffer (1X Tris buffer = 0.1 mol/L Tris-HCl, pH 7.6, 0.15 mol/L NaCl, and 0.05% Tween-20) at 46°C for 1 h, followed by incubation with streptavidin-Alexa 647 (Molecular Probes) at room temperature for 30 min in the dark. Arrays were then washed in 1X Tris buffer twice (5 min each), followed by a rinse in 0.05% Tween-20. Slides were then dried by centrifugation, kept in the dark, and scanned on an Axon GenePix Scanner using CodeLink Expression Scanning Software, and images were analyzed using CodeLink Expression Analysis Software. Expression values were globally normalized to the median expression value of the whole array spots.

    Real-time RT-PCR analysis. For independent verification of the results of the microarray analysis, real-time RT-PCR analysis of selected genes (Table 1) was performed using the SYBR Green method and the GeneAmp® 5700 Sequence Detection System (Applied Biosystems), as described (26). Primer pairs for rat genes were designed with Primer Express Software Version 1.5 (Applied Biosystems) and checked for sequence homology against known genes using a BLAST search (Table 1). Complementary DNA was synthesized from 10 µg total RNA isolated from rat abdominal adipose tissue, as described (27). Newly synthesized cDNA (150 ng) was used for PCR reactions. To ensure specific amplification, various negative controls (i.e., water only, reaction without primers, and templates derived without reverse transcriptase) were included in the PCR reaction. The specific location of the primers in the cDNA sequences and sizes of the amplified products are indicated in Table 2. Values for cycle threshold (C_T), the value of the cycle at which the fluorescence achieves a predetermined threshold, were determined using the Applied Biosystems Software. The C_T values were analyzed using the Generalized Estimating Equations model (28) and the PROC GENMOD procedure of the Statistical Analysis System. An exchangeable correlation structure was assumed for correlated observations within each animal sample (28). The model included the C_T value for carnitine palmitoyltransferase (CPT)-II as an offset to correct for differences in cDNA input into PCR reactions. Expression levels for the real-time RT-PCR analysis were calculated using a formula of 2^x–y, where x is the normalized C_T value obtained from alanine-treated rats for each primer set and y is the normalized C_T value obtained from arginine-treated rats, as suggested by Applied Biosystems.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Intake of food, energy and water. Food, water, and gross energy consumption over the 10-wk period of the study did not differ (P > 0.05) between alanine- and arginine-treated ZDF rats (Table 2). Intakes of all nutrients (including alanine and arginine) from the enteral diet, as well as the nitrogen provision from drinking water did not differ between the 2 groups of rats. In arginine-supplemented ZDF rats, arginine intake from drinking water was 4 times that from the enteral diet.

    Body weight. Initial body weights (at 9 wk of age) were 340.0 ± 3.6 and 341.3 ± 6.6 g, and final body weights (at 19 wk of age) were 407.7 ± 13.2 and 341.7 ± 8.5 g for alanine- and arginine-treated ZDF rats, respectively. The analysis of body weight changes during the 10-wk arginine supplementation period indicates a nonzero growth slope (age) (P < 0.01) and nonzero quadratic growth curvature (age²) (P < 0.01). Growth rates differed (P < 0.05) between alanine- and arginine-treated ZDF rats, with the control rats growing faster (P < 0.05) by 0.72 ± 0.12 g/d. Compared with alanine-treated rats, arginine-supplemented ZDF rats had 6.3, 10, and 16% lower (P < 0.05) body weights at wk 4, 7, and 10, respectively, after the treatment initiation (Fig. 1).

View larger version (23K): [in this window] [in a new window]   FIGURE 1 Body weights of ZDF rats. ZDF rats (9 wk old) were randomly assigned to receive drinking water containing either 1.51% L-arginine-HCl or 2.55% L-alanine (isonitrogenous control) for 10 wk. Data are means ± SEM, n = 6. *P < 0.05 vs. the alanine-treated rats.

    NO synthesis, substrate oxidation, and glycerol release by adipose tissues. Abdominal and epididymal adipose tissues of ZDF rats produced NO, oxidized glucose, and octanoate, and released glycerol (an indicator of lipolysis) (Table 6). Dietary arginine supplementation enhanced (P < 0.01) NO production (71–85%), the oxidation of glucose (34–36%) and octanoate (40–43%), and glycerol release (22–24%) in the adipose tissues.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Oral administration of arginine (1.25% in drinking water) was highly effective in enhancing serum levels of arginine (Table 4). The amount of supplemental arginine via drinking water was 4 times that via the enteral diet, and was well tolerated by all ZDF rats. No clinical side effects were noted in arginine-treated ZDF rats throughout the study, as reported for mice receiving oral administration of arginine (6% in drinking water) (35). Serum levels of histidine, lysine, and BCAA did not differ between control and arginine-treated ZDF rats, as reported previously for streptozotocin-diabetic rats (20). Taken together, these results indicate a lack of antagonism in the intestinal absorption of basic amino acids and of interference in intestinal absorption of neutral amino acids in response to oral arginine administration. Serum levels of both NO_x and citrulline (a coproduct of NO synthase) were increased in arginine-supplemented ZDF rats (Table 4), suggesting an increase in systemic NO production in response to the arginine treatment. The reduced serum levels of asymmetric dimethylarginine and N^G-monomethylarginine (inhibitors of NO synthase) as well as homocysteine (an oxidant) (Table 5) may contribute to an increase in NO production and bioavailability in the body (36).

An interesting observation from the present work is that dietary arginine supplementation increased the proportional weight of skeletal muscle, heart, and brain in ZDF rats (Table 3). This is likely beneficial for obese and diabetic patients whose muscle mass is relatively reduced (5). It is not known whether arginine treatment might affect protein turnover in these tissues. However, a recent study reported that enhancing arginine availability stimulated muscle protein synthesis without affecting muscle proteolysis in rabbits (37). The net result is an increase in muscle mass.

A novel, exciting finding of this study is that arginine supplementation markedly reduced body weight (Fig. 1) as well as the weights of abdominal and epididymal adipose tissues (Table 3). This is consistent with an increase in the oxidation of both glucose and fatty acid in adipose tissue of arginine-treated ZDF rats (Table 6), an indication of an overall increase in energy expenditure rather than a specific effect on lipid metabolism. A decrease in body fat mass is consistent with a reduction in serum levels of leptin (a hormone produced almost exclusively by adipocytes) in arginine-treated ZDF rats (Table 5). To provide a molecular basis for the antiobesity action of arginine, we conducted a study to determine gene expression in the abdominal adipose tissue of ZDF rats using microarray analysis. Our results indicated that expressions of NOS-1, HO-3, AMPPK, and PGC-1 were markedly increased in the adipose tissue of arginine-treated ZDF rats (Table 7). It is likely that increases in mRNA levels for these genes will translate into increases in protein expression, but this must be determined experimentally.

As noted above, PGC-1 is a master regulator of oxidative phosphorylation and mitochondrial biogenesis (8,9). Thus, the PGC-1 gene is associated with glucose metabolism in type-II diabetic patients (38), and its expression is reduced in insulin-sensitive tissues of type-II diabetic patients (39,40). In addition, AMPPK triggers the oxidation of energy substrates in skeletal muscle, heart, liver, and adipose tissue by reducing the availability of malonyl-CoA (an inhibitor of CPT-I) via an inhibition of acetyl-CoA carboxylase and an activation of malonyl-CoA decarboxylase (41,42). Furthermore, cGMP stimulates the mitochondrial oxidation of acetyl-CoA in animal cells via an inhibition of acetyl-CoA carboxylase (7). cGMP also enhances lipolysis in adipocytes via the phosphorylation of hormone-sensitive lipase and perilipin (43). Notably, NO enhances PGC-1 expression and activates AMPPK (10), whereas both NO and carbon monoxide (CO, a product of heme oxygenase) activate guanylyl cyclase to generate cGMP (44–46). Thus, the increases in expression of the genes for NOS-1, HO-3, AMPPK, and PGC-1 in adipose tissue would be expected to coordinately promote mitochondrial oxidation of energy substrates, thereby decreasing the availability of long-chain fatty acyl-CoA for triglyceride synthesis and of acetyl-CoA for fatty acid synthesis (Fig. 2). This would result in an overall increase in the oxidation of both glucose and fatty acids while possibly decreasing fat deposition. The outcome is to reduce fat mass in the body.

View larger version (26K): [in this window] [in a new window]   FIGURE 2 A proposed mechanism for the role of dietary arginine supplementation in enhancing substrate oxidation. Enzymes that catalyze the indicated reactions are as follows: 1) Krebs cycle enzymes; 2) nitric oxide synthase; 3) guanylyl cyclase; 4) heme oxygenase; 5) pyruvate dehydrogenase; 6) long-chain fatty acyl-CoA synthase; 7) enzymes of glycolysis. CPT-I is required for the transport of long-chain fatty acyl-CoA from the cytosol into mitochondria for ß-oxidation. NO, produced from L-arginine by NO synthase, enhances expression of PGC-1 (a master regulator of oxidative phosphorylation and mitochondrial biogenesis). Both NO and CO (a product of heme oxygenase) activate guanylyl cyclase to generate cGMP, which stimulates the mitochondrial oxidation of acetyl-CoA and lipolysis. The symbol "+" denotes stimulation.

Dietary arginine supplementation represents a potentially novel and useful strategy for the management of obesity and diabetes (47–49). Arginine is a stable amino acid in an aqueous solution and under sterilization conditions (e.g., high temperature and high pressure) (49). In addition, arginine is not toxic, and its administration is generally safe for both humans and animals (35,50,51). In type-II diabetic patients, the pancreas is exhausted from overproduction of insulin to overcome its resistance in tissues, and most clinicians do not use drugs or agents that stimulate insulin secretion to treat this type of diabetes (52). In contrast to streptozotocin-induced diabetic rats (20), dietary supplementation of arginine had no effect on plasma insulin levels in ZDF rats (Table 5). Similarly, arginine treatment did not increase plasma concentration of insulin in type-II diabetic patients (53). Arginine supplementation may become a promising solution to reduce obesity, improve insulin sensitivity, and improve health in type-II diabetic subjects. In obese or overweight subjects, even a modest (6–8%) decrease in body weight will yield substantial health benefits (4). Thus, our findings will not only fundamentally advance the field of arginine-NO research, but will also aid in the development of novel therapeutics with the potential to ameliorate the worldwide obesity epidemic and its associated metabolic abnormalities.

In conclusion, results of this study indicate that dietary arginine supplementation, even if started in adult life, selectively reduced the mass of adipose tissue and promoted the loss of body weight in ZDF rats. The arginine treatment enhanced the expression of 4 key genes responsible for fatty acid and glucose oxidation in adipose tissue and reduced serum levels of glucose, FFA, triglycerides, homocysteine, and leptin. Thus, arginine is a potentially novel antiobesity amino acid and its dietary supplementation may be beneficial for the treatment of obese and diabetic patients.

	ACKNOWLEDGMENTS

 
We thank Jon Self, Wene Yan, Katherine Kelly, and Scott Jobgen for excellent technical assistance as well as Susan K. Fried, Stephen B. Smith, and Malcolm Watford for helpful discussion.

	FOOTNOTES

 
¹ Supported in part by grants from American Heart Association #0255878Y, Juvenile Diabetes Research Foundation #1–2002-228, National Institutes of Health/National Cancer Institute #R25 CA90301 and CA57030, NIH/National Heart, Lung, and Blood Institute R21 HL077401–01, and National Institute of Environmental Health Sciences #P30-ES09106.

³ Abbreviations used: AMPPK, AMP-activated protein kinase; CPT, carnitine palmitoyltransferase; C_T, cycle threshold; FDR, false discovery rate; GLUT, glucose transporters; HO, heme oxygenase; NOS, nitric oxide synthase; NO_x, oxidation products of nitric oxide; ODC, ornithine decarboxylase; PGC-1, peroxisome proliferator-activated receptor coactivator-1; ZDF, Zucker diabetic fatty.

Manuscript received 15 November 2004. Initial review completed 4 January 2005. Revision accepted 16 January 2005.

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