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, 289 (14), 9710-29

The Molecular and Metabolic Influence of Long Term Agmatine Consumption

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The Molecular and Metabolic Influence of Long Term Agmatine Consumption

Itzhak Nissim et al. J Biol Chem.

Abstract

Agmatine (AGM), a product of arginine decarboxylation, influences multiple physiologic and metabolic functions. However, the mechanism(s) of action, the impact on whole body gene expression and metabolic pathways, and the potential benefits and risks of long term AGM consumption are still a mystery. Here, we scrutinized the impact of AGM on whole body metabolic profiling and gene expression and assessed a plausible mechanism(s) of AGM action. Studies were performed in rats fed a high fat diet or standard chow. AGM was added to drinking water for 4 or 8 weeks. We used (13)C or (15)N tracers to assess metabolic reactions and fluxes and real time quantitative PCR to determine gene expression. The results demonstrate that AGM elevated the synthesis and tissue level of cAMP. Subsequently, AGM had a widespread impact on gene expression and metabolic profiling including (a) activation of peroxisomal proliferator-activated receptor-α and its coactivator, PGC1α, and (b) increased expression of peroxisomal proliferator-activated receptor-γ and genes regulating thermogenesis, gluconeogenesis, and carnitine biosynthesis and transport. The changes in gene expression were coupled with improved tissue and systemic levels of carnitine and short chain acylcarnitine, increased β-oxidation but diminished incomplete fatty acid oxidation, decreased fat but increased protein mass, and increased hepatic ureagenesis and gluconeogenesis but decreased glycolysis. These metabolic changes were coupled with reduced weight gain and a curtailment of the hormonal and metabolic derangements associated with high fat diet-induced obesity. The findings suggest that AGM elevated the synthesis and levels of cAMP, thereby mimicking the effects of caloric restriction with respect to metabolic reprogramming.

Keywords: Adenylate Cyclase (Adenylyl Cyclase); Carbohydrate Metabolism; Carnitine; Cyclic AMP (cAMP); Fatty Acid Oxidation; Insulin Resistance; Liver; Metabolic Syndrome.

Figures

FIGURE 1.
FIGURE 1.
Agmatine consumption, calorie intake, and body weight during the course of the study. a represents the daily consumption of agmatine. b represents the amount of agmatine in the liver or kidney at the end of 4 or 8 weeks of consumption as in a. c represents the daily caloric intake. d represents the changes in body weight during the course of SD or HFD with or without agmatine. Error bars represent means ± S.D. of six rats between 0 and 32 days and four rats in each experimental group between 32 and 56 days.
FIGURE 2.
FIGURE 2.
Plasma hormone and metabolite levels. a–i, measurements were performed in plasma of rats after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. Error bars represent mean ± S.D. (n = 5–6 rats; for glucagon or glucagon/insulin ratio, n = 4 rats). P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 3.
FIGURE 3.
Protein and fat mass in the liver and muscle. a–h, measurements were performed in freeze clamped tissue obtained from rats after 4 weeks on SD, SD + AGM, HFD (FD), or HFD + AGM. Error bars represent mean ± S.D. (n = 5–6 rats). P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 4.
FIGURE 4.
A representative 13C NMR analysis of hepatic lipids. 13C NMR spectra of hepatic lipid extract from rats after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM are shown. These spectra demonstrate a remarkable reduction in TG and fatty acids in the liver of rats that received AGM. Spectra were obtained using a Bruker Avance IIITM 400 wide bore and TopspinTM 3.0 software. The chemical shifts were determined relative to the resonance of trimethylsilylpropionic acid. Peak assignments are as follows: TG C2′, carbon 2 of glycerol in TG; TG C13′, carbons 1 and 3 of glycerol in TG; -N(CH3)3, trimethylammonium group in phosphatidylcholine and sphingomyelin; C2, carbon α to carbonyl in fatty acids; ω-2, carbon β to end chain CH3 in fatty acids; (CH2)n, bulk of CH2 groups in fatty acids; ω-1, carbon α to end chain CH3 in fatty acids; ω, end chain CH3 in fatty acids.
FIGURE 5.
FIGURE 5.
The expression of UCP2 and UCP3 genes. a–f, mRNA expression was determined in the liver, muscle, and eWAT by RT-PCR with GAPDH as an internal control (31). Levels of mRNA (in arbitrary units (A.U)) were calculated relative to SD. Error bars represent means ± S.D. (n = 4–6 rats). P1, significantly different from control (SD); P2, significantly different from HFD (FD).
FIGURE 6.
FIGURE 6.
Tandem mass spectrometry-based analysis of the plasma Car/AcylCar profiling after 4 weeks of agmatine consumption. a–n, measurements were performed in plasma of rats after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. Error bars represent means ± S.D. (n = 5–6 rats in each study group). P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 7.
FIGURE 7.
Impact of AGM on fat oxidation and generation of C2-acylcarnitine and KB. The top panel illustrates the potential sources of C2-acylcarnitine and KB that are expected to dilute the 13C enrichment (molar percent enrichment (MPE)) of C2-acylcarnitine and KB derived from the injected [U-13C]acetate. As unlabeled acetyl-CoA forms from various sources, a greater dilution of 13C-labeled metabolites occurs. GC-MS measurements were performed in plasma, muscle, and liver after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. 13C2-acylcarnitine is the M2 isotopomer (contains two carbons labeled with 13C). ΣM2,M4 13C-labeled ketone bodies represent the sum of M2 and M4 isotopomers of KB. Error bars represent means ± S.D. (n = 5–6 in each experimental group). The bottom panels represent experiments with isolated hepatocytes obtained from rats given SD or SD + AGM for 4 weeks. Incubations were performed with [U-13C]palmitate. Error bars represent means ± S.D. of three to four independent experiments with hepatocytes obtained from three to four rats in each group. P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 8.
FIGURE 8.
The impact of AGM on gene expression and enzyme activity of carnitine biosynthesis. The top panel represents the various steps in Car biosynthesis and the genes involved. Carnitine biosynthesis consists of four distinct enzymatic reactions. The TML generated from proteolysis is oxidized by TML dioxygenase (TMLD), 3-hydroxy-TML aldolase (HTMLA), and 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) to form γ-butyrobetaine. The last step is hydroxylation of γ-butyrobetaine by γ-BBD to form carnitine (24). Measurements were performed in rat tissues after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. For determination of gene expression, we used RT-PCR with GAPDH as an internal control. Levels of mRNA (arbitrary units (A.U)) were calculated relative to SD. Panel g represents the ex vivo determination of γ-BBD activity using a separate portion of the freeze clamped liver that was also used for determination of gene expression (as in a–c). Panel h represents the activity of γ-BBD in isolated hepatocytes obtained from rats given SD or SD + AGM for 4 weeks. For in vivo measurements, error bars represent means ± S.D. of four to six rats in each experimental group (a–f). P1, significantly different from control (SD); P2, significantly different from HFD. In experiments with isolated hepatocytes, error bars represent means ± S.D. of three to four independent incubations of hepatocytes obtained from three to four rats. P1, significantly different from control (SD).
FIGURE 9.
FIGURE 9.
Agmatine up-regulates the genes responsible for carnitine transport in the body. We determined gene expression of the organic cation transporters (OCTN1 and OCTN2) in the liver (a and b), muscle (c and d), and kidney (e and f) by RT-PCR using GAPDH as an internal control. Measurements were performed in rats after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. Levels of mRNA (arbitrary units (A.U)) were calculated relative to SD. Error bars represent means ± S.D. of four to six rats in each group. P1, significantly different from control (SD); P2, significantly different from HFD.
FIGURE 10.
FIGURE 10.
The influence on hepatic carbohydrate and organic acids metabolism. The top panel illustrates the potential coupling between the hepatic acetyl-CoA pool and the metabolism of lactate, pyruvate, the TCA cycle, gluconeogenesis, and glycolysis. Mal, malate; Cit, citrate; Oxa, oxaloacetate; α-Kg, α-ketoglutarate; Lac, lactate; LDH, lactate dehydrogenase; Pyr, pyruvate; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; GDH, glutamate dehydrogenase; G6P, glucose 6-phosphate. a–e represent the levels of total glucose and organic acids in the liver of rats after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. Metabolite levels were measured in liver extracts prepared from rats following the 4 week dietary regimen as indicated. Metabolite levels are means ± S.D. of five to six rats in each group. f–l represent the metabolism of 13C-labeled precursors in isolated hepatocytes obtained from rats given either SD or SD + AGM for 4 weeks. All incubations were carried out for 60 min as described under “Materials and Methods.” f represents the generation of 13CO2 from [U-13C]glucose via glycolysis. g represents the generation of 13C-labeled lactate from [U-13C]glucose via glycolysis. h represents the sum of 13C-labeled glucose isotopomers production from [1-13C]-labeled pyruvate and lactate via gluconeogenesis. i represents the release of 13CO2 from [1-13C]pyruvate via the pyruvate dehydrogenase reaction. j represents the generation of M1 isotopomer of aspartate from [1-13C]pyruvate via anaplerosis. k represents the release of 13CO2 from [3-13C]pyruvate via the TCA cycle. l represents the generation of the M2 isotopomer (labeled at two carbons) of glutamate from [3-13C]pyruvate via cataplerosis. Error bars in experiments with isolated hepatocytes represent means ± S.D. of three to four independent experiments with hepatocytes obtained from three to four rats in each group. P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 11.
FIGURE 11.
The impact of AGM on waste nitrogen removal via hepatic ureagenesis. The top panel illustrates the coupling between FAO, the acetyl-CoA pool, the TCA cycle, and the synthesis of NAG via N-acetylglutamate synthetase (NAGS) and thereby activation of ureagenesis. Also shown are the generation of ammonia via phosphate-dependent glutaminase (PDG) and the reductive amination of α-ketoglutarate (α-kg) via glutamate dehydrogenase (GDH). NAG activates the carbamoyl-phosphate synthetase-I (CPS-1) to form carbamoyl phosphate (CP) and its conversion into citrulline via ornithine transcarbamylase (OTC). a–c represent the levels of plasma ammonia, total blood urea, and synthesis of 15N-labeled urea (sum of urea containing one and two 15N atoms) from 15NH4Cl injected in rats after 4 weeks of SD, SD + AGM, HFD, or HFD + AGM (details are under “Materials and Methods”). d and e represent the total levels of NAG and glutamate in the liver of the same rats. f represents the generation of 15N-labeled glutamate in experiments with isolated hepatocytes incubated with 15NH4Cl. g and h represent the generation of 13C-labeled NAG at two carbons (M2) and the total level of NAG following incubation of isolated hepatocytes with [U-13C]palmitate. i represents the generation of 15N-labeled urea in experiments with isolated hepatocytes incubated with 15NH4Cl. All incubations were carried out for 60 min as described under “Materials and Methods.” In a–e, error bars represent means ± S.D. of five to six rats in each experimental group. In f–i, error bars represent means ± S.D. of three to four independent experiments with hepatocytes obtained from three to four rats in each group. P1, significantly different from SD; P2, significantly different from HFD.
FIGURE 12.
FIGURE 12.
Agmatine up-regulates adenylyl cyclase expression, synthesis and levels of cAMP, and the resulting expression of downstream target genes. Measurements were performed in rat tissues after 4 weeks on SD, SD + AGM, HFD, or HFD + AGM. In the liver, AGM increased the expression of ADCY5 with no or little effect on ADCY6 (a and b), resulting in increased activity of ADCY (c) as measured in plasma membranes prepared from liver homogenates (a portion of the same livers used for mRNA analysis) and thereby significantly increased [cAMP] both in SD + AGM and HFD + AGM study groups (d). The elevated cellular [cAMP] is coupled with increased expression of CREB, PPARα, and PGC1α as indicated in e, f, and g, respectively. In the eWAT, AGM increased expression of ADCY5 and ADCY6 (h and i), augmented the cAMP level (j), and thereby significantly increased expression of CREB and PPARγ (k and l) with only a modest increase in PPARα expression (m). Gene expression was determined by RT-PCR using GAPDH as an internal control. Levels of mRNA (arbitrary units (A.U)) were calculated relative to SD. Error bars represent means ± S.D. of four to six rats in each group. P1, significantly different from control (SD); P2, significantly different from HFD.
FIGURE 13.
FIGURE 13.
A schematic representation of the impact of agmatine on the molecular and metabolic pathways. The current findings indicate that AGM elevates the synthesis and levels of cAMP (Fig. 12) and subsequently may activate the cAMP-PKA pathway. The latter initiates a molecular and metabolic chain reaction including phosphorylation and activation of CREB, PGC1α, and PPARα. The up-regulation of PPARα resulted in activation of downstream target genes responsible for Car synthesis and transport (Figs. 8 and 9) and hence augmented the synthesis of Car and its level in plasma (Fig. 6) and tissues (Table 2). The increased synthesis of Car together with increased expression of CREB and PGC1α led to increased β-oxidation (Fig. 7) and increased fluxes through pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), the TCA cycle, and gluconeogenesis but decreased glycolysis (Fig. 10). Simultaneously, the up-regulation of UCPs (Fig. 5) is expected to elevate the metabolic rate. The latter, when coupled with elevated expression of PPARγ in adipose tissue (Fig. 12), resulted in higher fat oxidation as well as decreased fat mass (Figs. 3 and 4), decreased body weight gain (Fig. 1), and thereby improved insulin sensitivity (Fig. 2). In addition, the products of β-oxidation, such as acetyl-CoA together with the augmented cataplerosis and generation of glutamate through the TCA cycle, resulted in augmented synthesis of NAG, stimulation of ureagenesis, and removal of waste nitrogen from the body (Fig. 11). These coordinated changes in gene expression and metabolic pathways and thereby the AGM-induced metabolic reprogramming suggest that AGM may lessen the so-called metabolic syndrome, i.e. the metabolic malfunctions associated with HFD-induced obesity. ↑ indicates stimulation or improvement, and ↓ indicates decrease.

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