Continuous oxidative stress due to activation of polyamine catabolism accelerates aging and protects against hepatotoxic insults.

Enhanced polyamine catabolism via polyamine acetylation-oxidation elevates the oxidative stress in an organism due to increased production of reactive oxygen species (ROS). We studied a transgenic mouse line overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase (SSAT) that is characterized by increased putrescine and decreased spermidine and spermine pools. In order to protect the mice from the chronic oxidative stress produced by the activation of polyamine catabolism, the hepatic expression of the transcription factor p53 was found threefold elevated in the transgenic mice. In addition, the prolonged activation of p53 accelerated the aging of transgenic mice and reduced their lifespan (50%). Aging was associated with decreased antioxidant enzyme activities. In the transgenic mice the activities of catalase and Cu, Zn-superoxide dismutase (SOD) were 42 and 23% reduced respectively, while the expression of CYP450 2E1 was 60% decreased and oxidative stress measured as protein carbonyl content was tenfold elevated. In the transgenic mice, the age-related repression of the different antioxidant enzymes served as a protection against the hepatotoxic effects of carbon tetrachloride and thioacetamide.

The role of spermidine/spermine N1-acetyltransferase in endotoxin-induced acute kidney injury.

The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.

Proteomic analysis of livers from a transgenic mouse line with activated polyamine catabolism.

We have generated a transgenic mouse line that over expresses the rate-controlling enzyme of the polyamine catabolism, spermidine/spermine N (1)-acetyltransferase, under the control of a heavy metal inducible promoter. This line is characterized by a notable increase in SSAT activity in liver, pancreas and kidneys and a moderate increase in the rest of the tissues. SSAT induction results in an enhanced polyamine catabolism manifested as a depletion of spermidine and spermine and an overaccumulation of putrescine in all tissues. To study how the activation of polyamine catabolism affects other metabolic pathways, protein expression pattern of the livers of transgenic animals was analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. A total of 23 proteins were shown to be differentially expressed in the transgenic from the wild-type animals. Many of the identified proteins showed expression patterns associated with polyamine catabolism activation. However, the expression pattern of other proteins, such as repression of GST pi and selenium-binding protein 2 and 60 kDa heat-shock protein, could be explained by the overexpression of peroxisome proliferator-activated receptor gamma co-activator 1alpha in response to depleted ATP pools. The activation of the latter proteins is thought to lead to the improved insulin sensitivity seen in the MT-SSAT animals.

Cutaneous application of alpha-methylspermidine activates the growth of resting hair follicles in mice.

Recent studies using transgenic animals have revealed a crucial role for polyamines in the development and the growth of skin and hair follicles. In mammals, the growth of hair is characterized by three main cyclic phases of transformation, including a rapid growth phase (anagen), an apoptosis-driven regression phase (catagen) and a relatively quiescent resting phase (telogen). The polyamine pool during the anagen phase is higher than in telogen and catagen phases. In this study, we used alpha-methylspermidine, a metabolically stable polyamine analog, to artificially elevate the polyamine pool during telogen. This manipulation was sufficient to induce hair growth in telogen phase mice after 2 weeks of daily topical application. The application site was characterized by typical features of anagen, such as pigmentation, growing hair follicles, proliferation of follicular keratinocytes and upregulation of beta-catenin. The analog penetrated the protective epidermal layer of the skin and could be detected in dermis. The natural polyamines were partially replaced by the analog in the application site. However, the combined pool of natural spermidine and alpha-methylspermidine exceeded the physiological spermidine pool in telogen phase skin. These results highlight the role of polyamines in hair cycle regulation and show that it is possible to control the process of hair growth using physiologically stable polyamine analogs.

Activated polyamine catabolism leads to low cholesterol levels by enhancing bile acid synthesis.

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) have significantly reduced plasma total cholesterol levels. In our study, we show that low cholesterol levels were attributable to enhanced bile acid synthesis in combination with reduced cholesterol absorption. Hepatic cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme catalyzing the conversion of cholesterol to bile acids, plays an important role in the removal of excess cholesterol from the body. We suggest that by reducing activity of Akt activated polyamine catabolism increased the stability and activity of peroxisome proliferator-activated receptor gamma co-activator 1alpha, the critical activator of CYP7A1. This is supported by our finding that the treatment with SSAT activator, N (1) ,N(11)-diethylnorspermine, reduced significantly the amount of phosphorylated (active) Akt in HepG2 cells. In summary, activated-polyamine catabolism is a novel mechanism to regulate bile acid synthesis. Therefore, polyamine catabolism could be a potential therapeutic target to control hepatic CYP7A1 expression.

Spermidine is indispensable in differentiation of 3T3-L1 fibroblasts to adipocytes.

Impaired adipogenesis has been shown to predispose to disturbed adipocyte function and development of metabolic abnormalities. Previous studies indicate that polyamines are essential in the adipogenesis in 3T3-L1 fibroblasts. However, the specific roles of individual polyamines during adipogenesis have remained ambiguous as the natural polyamines are readily interconvertible inside the cells. Here, we have defined the roles of spermidine and spermine in adipogenesis of 3T3-L1 cells by using (S')- and (R')- isomers of alpha-methylspermidine and (S,S')-, (R,S)- and (R,R')-diastereomers of alpha,omega-bismethylspermine. Polyamine depletion caused by alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented adipocyte differentiation by suppressing the expression of its key regulators, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Adipogenesis was restored by supplementation of methylspermidine isomers but not of bismethylspermine diastereomers. Although both spermidine analogues supported adipocyte differentiation only (S)-methylspermidine was able to fully support cell growth after extended treatment with alpha-DFMO. The distinction between the spermidine analogues in maintaining growth was found to be in their different capability to maintain functional hypusine synthesis. However, the differential ability of spermidine analogues to support hypusine synthesis did not correlate with their ability to support differentiation. Our results show that spermidine, but not spermine, is essential for adipogenesis and that the requirement of spermidine for adipogenesis is not strictly associated with hypusine modification. The involvement of polyamines in the regulation of adipogenesis may offer a potential application for the treatment of dysfunctional adipocytes in patients with obesity and metabolic syndrome.

Divergent regulation of the key enzymes of polyamine metabolism by chiral alpha-methylated polyamine analogues.

The natural polyamines are ubiquitous multifunctional organic cations which play important roles in regulating cellular proliferation and survival. Here we present a novel approach to investigating polyamine functions by using optical isomers of MeSpd (alpha-methylspermidine) and Me2Spm (alpha,omega-bismethylspermine), metabolically stable functional mimetics of natural polyamines. We studied the ability of MeSpd and Me2Spm to alter the normal polyamine regulation pathways at the level of polyamine uptake and the major control mechanisms known to affect the key polyamine metabolic enzymes. These include: (i) ODC (ornithine decarboxylase), which catalyses the rate-limiting step of polyamine synthesis; (ii) ODC antizyme, an inhibitor of ODC and polyamine uptake; (iii) SSAT (spermidine/spermine N1-acetyltransferase), the major polyamine catabolic enzyme; and (iv) AdoMetDC (S-adenosyl-L-methionine decarboxylase), which is required for the conversion of putrescine into spermidine, and spermidine into spermine. We show that the stereoisomers differ in their cellular uptake and ability to downregulate ODC and AdoMetDC, and to induce SSAT. These effects are mediated by the ability of the enantiomers to induce +1 ribosomal frameshifting on ODC antizyme mRNA, to suppress the translation of AdoMetDC uORF (upstream open reading frame) and to regulate the alternative splicing of SSAT pre-mRNA. The unique effects of chiral polyamine analogues on polyamine metabolism may offer novel possibilities for studying the physiological functions, control mechanisms, and targets of the natural polyamines, as well as advance therapeutic drug development in cancer and other human health-related issues.

Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts.

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT-EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N(1),N(11)-diethylnorspermine), CPENSpm (N(1)-ethyl-N(11)-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N(1)-ethyl-N(11)-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT-EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT-PCR (reverse transcription-PCR) results suggested that the analogue-affected regulation of SSAT-EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

Spermidine/spermine-N1-acetyltransferase ablation protects against liver and kidney ischemia-reperfusion injury in mice.

Expression of spermine/spermidine-N1-acetyltransferase (SSAT), the rate-limiting enzyme of polyamine backconversion cascade, increases after ischemia-reperfusion injuries (IRI). We hypothesized that SSAT plays an important role in the mediation of IRI. To test our hypothesis, wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice were subjected to liver or kidney IRI by ligation of hepatic or renal arteries. The liver and kidney content of putrescine (Put), a downstream by-product of SSAT activity, increased in SSAT-wt animals but not in SSAT-ko animals after IRI, indicating that polyamine backconversion is not functional in SSAT-deficient mice. When subjected to hepatic IRI, SSAT-ko mice were significantly protected against liver damage compared with SSAT-wt mice. Similarly, SSAT-ko animals subjected to renal IRI showed significantly greater protection against damage to kidney tubules than SSAT-wt mice. These studies indicate that SSAT-deficient animals are protected against IRI and suggest that SSAT is an important mediator of the tissue damage in IRI.

Quantitative determination of underivatized polyamines by using isotope dilution RP-LC-ESI-MS/MS.

A rapid, sensitive and selective method using LC-MS/MS was developed and validated for the simultaneous quantitative determination of five polyamines N1,N12-diethylspermine (DESpm), N-ethylspermine (EtSpm), N1-ethylspermidine (EtSpd), spermidine (Spd) and N1-ethyl-1,3-diaminopropane (EtDAP) without any derivatization steps. The LC-MS/MS system was operated using the positive electrospray ionization (ESI) mode. The chromatographic separation only took 10min and was performed on a reversed phase C18 column with 0.1% heptafluorobutyric acid as the ion-pairing agent and acetonitrile gradient. Stable, deuterium labelled internal reference compounds of the five analytes were included in the quantification. The lower limit of quantification for all of the five analytes was 0.03microM and the method was linear for DESpm, EtSpd, Spd and EtDAP over the range of 0.03-60microM and for EtSpm over the range of 0.03-30microM. Correlation coefficients (R2) were always >0.995 for all the analytes. The precision of the overall method ranged from 0.2 to 9.7% as intra-day variability and from 0.9 to 6.8% as inter-day variability. The intra-day and inter-day accuracy of the assay ranged between 87.6-109.8% and 89.6-106.6%, respectively. The method has been applied successfully to quantify metabolites of DESpm as a substrate for recombinant human polyamine oxidase.

Downregulation of PPARs and SREBP by acyl-CoA-binding protein overexpression in transgenic rats.

Acyl-CoA-binding protein (ACBP) acts as an acyl-CoA pool former, transporter, and regulator of gene transcription in vitro. We created a transgenic rat line overexpressing ACBP, as the physiological relevance of ACBP in lipid metabolism is unclear. Transgenic rats revealed increased levels of ACBP and significantly elevated acyl-CoA tissue levels while there was no effect on plasma triglyceride, cholesterol, or serum-free fatty acid levels. Metabolic regulators like peroxisome proliferator-activated receptors (PPARgamma, PPARdelta) and sterol regulatory element-binding protein-1 (SREBP-1) messenger RNA levels were significantly reduced (by 23-82%) in liver and adipose tissue of fed transgenic rats, whereas adenosine monophosphate-activated protein kinase (AMPK) protein levels were increased (by 60%). Fasting abolished PPAR downregulation in liver and caused an upregulation in adipose tissue. Administration of AMPK inhibitor reversed SREBP-1 but did not affect PPAR regulation. In conclusion, ACBP acts as an acyl-CoA pool former in transgenic rats and regulates lipid metabolism via SREBP-1 and PPAR regulation. Reduction of SREBP-1 is mediated via increased AMPK levels, whereas regulation of PPARs seems to be mediated by an AMPK-independent mechanism. ACBP itself is a target gene for both transcription factors demonstrating important feedback loops.

Novel CoA-polyamine conjugates for effective inhibition of spermine/spermidine-N1-acetyltransferase.

New mimics of the transition state of spermine/spermidine-N(1)-acetyltransferase reaction were prepared starting from aminooxy analogues of spermidine or spermine and SH-CoA. The activity depended on the structure of polyamine fragment of the conjugate and best of the synthesized compounds were active at micromolar concentrations.

Analysis of underivatized polyamines by reversed phase liquid chromatography with electrospray tandem mass spectrometry.

A reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometric method (RP-LC-ESI-MS/MS) was developed to separate and detect polyamines with minimal sample pre-treatment and without any derivatization. Prior to MS/MS analysis, a complete chromatographic separation of polyamines was achieved by a linear gradient elution using heptafluorobutyric acid as a volatile ion-pair modifier, and signal suppression was prevented by post-column addition of 75% propionic acid in isopropanol to the column flow. The developed method was successfully applied to the identification of metabolites formed from N(1),N(12)-diethylspermine in the reaction catalyzed by recombinant human polyamine oxidase and to the detection of eight different polyamines in a standard mixture.

Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis.

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.

Alpha-methylated polyamines as potential drugs and experimental tools in enzymology.

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.

Enhanced polyamine catabolism alters homeostatic control of white adipose tissue mass, energy expenditure, and glucose metabolism.

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.

Diazepam binding inhibitor overexpression in mice causes hydrocephalus, decreases plasticity in excitatory synapses and impairs hippocampus-dependent learning.

Diazepam binding inhibitor (DBI) and its processing products are endogenous modulators of GABAA and linked to various brain disorders ranging from anxiety and drug dependence to epilepsy. To investigate the physiological role of endogenously expressed DBI in the brain we created a transgenic mouse line overexpressing DBI gene. Transgenic mice had a 37x increased protein expression and immunohistochemistry showed excessive glial expression in the infragranular region of the dentate gyrus. Transgenic animals had significantly larger lateral ventricles and decreased plasticity of excitatory synapses without affecting either inhibitory or excitatory synaptic transmission. In behavioral tests transgenic animals had no differences in motor and exploratory activity, yet impaired hippocampus-dependent learning and memory. Overexpression did not cause anxiety or proconflict behavior, nor influenced kainic acid or pentylenetetrazole induced seizure activity. Our transgenic mouse line demonstrates that endogenously overexpressed DBI impairs hippocampus-dependent learning without anxiety or proconflict behavior.

Short and long-term effects of hVEGF-A(165) in Cre-activated transgenic mice.

We have generated a transgenic mouse where hVEGF-A(165) expression has been silenced with loxP-STOP fragment, and we used this model to study the effects of hVEGF-A(165) over-expression in mice after systemic adenovirus mediated Cre-gene transfer. Unlike previous conventional transgenic models, this model leads to the expression of hVEGF-A(165) in only a low number of cells in the target tissues in adult mice. Levels of hVEGF-A(165) expression were moderate and morphological changes were found mainly in the liver, showing typical signs of active angiogenesis. Most mice were healthy without any major consequences up to 18 months after the activation of hVEGF-A(165) expression. However, one mouse with a high plasma hVEGF-A(165) level died spontaneously because of bleeding into abdominal cavity and having liver hemangioma, haemorrhagic paratubarian cystic lesions and spleen peliosis. Also, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in control mice. We conclude that long-term uncontrolled hVEGF-A(165) expression in only a limited number of target cells in adult mice can be associated with pathological changes, including possible formation of malignant tumors and uncontrolled bleeding in target tissues. These findings have implications for the design of long-term clinical trials using hVEGF-A(165) gene and protein.

Mice with targeted disruption of spermidine/spermine N1-acetyltransferase gene maintain nearly normal tissue polyamine homeostasis but show signs of insulin resistance upon aging.

The N(1)-acetylation of spermidine or spermine by spermidine/spermine N(1)-acetyltransferase (SSAT) is the ratecontrolling enzymatic step in the polyamine catabolism. We have now generated SSAT knockout (SSAT-KO) mice, which confirmed our earlier results with SSATdeficient embryonic stem (ES) cells showing only slightly affected polyamine homeostasis, mainly manifested as an elevated molar ratio of spermidine to spermine in most tissues indicating the indispensability of SSAT for the spermidine backconversion. Contrary to SSAT deficient ES cells, polyamine pools in SSAT-KO mice remained almost unchanged in response to N(1),N(11)-diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polyamine pools in the wild-type animals and ES cells. Furthermore, SSATKO mice were more sensitive to the toxicity exerted by DENSPM in comparison with wild-type mice. The latter finding indicates that inducible SSAT plays an essential role in vivo in DENSPM treatmentevoked polyamine depletion, but a controversial role in toxicity of DENSPM. Surprisingly, liver polyamine pools were depleted similarly in wild-type and SSAT-KO mice in response to carbon tetrachloride treatment. Further characterization of SSAT knockout mice revealed insulin resistance at old age which supported the role of polyamine catabolism in glucose metabolism detected earlier with our SSAT overexpressing mice displaying enhanced basal metabolic rate, high insulin sensitivity and improved glucose tolerance. Therefore SSAT knockout mice might serve as a novel mouse model for type 2 diabetes.

Polyamine-regulated unproductive splicing and translation of spermidine/spermine N1-acetyltransferase.

Spermidine/spermine N1-acetyltransferase (SSAT), the rate-controlling enzyme in the interconversion of spermidine and spermine, is regulated by polyamines and their analogs at many levels of gene expression. Recently, SSAT pre-mRNA has been shown to undergo alternative splicing by inclusion of an exon that contains premature termination codons. In the present study, we show that alterations in the intracellular polyamine level resulted in a change in the relative abundance of SSAT transcripts. Addition of polyamines or their N-diethylated analogs reduced the amount of the variant transcript, whereas polyamine depletion by 2-difluoromethylornithine or MG-132 enhanced the exon inclusion. Experiments performed with protein synthesis inhibitors and siRNA-mediated down-regulation of Upf1 protein verified that the variant transcript was degraded by nonsense-mediated mRNA decay (NMD). Interestingly, several proteins have been shown to regulate their expression by alternative splicing-coupled NMD, termed regulated unproductive splicing and translation (RUST). Our present results suggest that in the case of SSAT, RUST is mediated by polyamines, and this system functions to fine-tune the polyamine metabolism.