Anne Uimari

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL‐4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL‐4‐dependent E‐cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL‐4‐induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1‐deficient macrophages display rather enhanced IL‐4‐induced polyamine production, suggesting that an Arg1‐independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS‐induced expression of several proinflammatory genes was increased significantly in polyamine‐depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL‐4‐induced expression of several AAM mediators, they inhibit the LPS‐mediated expression of proinflammatory genes in CAMs.

Animal disease models generated by genetic engineering of polyamine metabolism

The polyamines putrescine, spermidine and spermine are natural components of all living cells. Although their exact cellular functions are still largely unknown, a constant supply of these compounds is required for mammalian cell proliferation to occur. Studies with animals displaying genetically altered polyamine metabolism have shown that polymines are intimately involved in the development of diverse tumors, putrescine apparently has specific role in skin physiology and neuroprotection and the higher polyamines spermidine and spermine are required for the maintenance of pancreatic integrity and liver regeneration. In the absence of ongoing polyamine biosynthesis, murine embryogenesis does not proceed beyond the blastocyst stage. The last years have also witnessed the appearance of the first reports linking genetically altered polyamine metabolism to human diseases.

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 α‐methylspermidine and (S,S’)‐, (R,S)‐ and (R,R’)‐diastereomers of α,ω‐bismethylspermine. Polyamine depletion caused by α‐difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented adipocyte differentiation by suppressing the expression of its key regulators, peroxisome proliferator‐activated receptor γ and CCAAT/enhancer binding protein α. 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 α‐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.

Oxidative stress and inflammation in the pathogenesis of activated polyamine catabolism-induced acute pancreatitis

Summary.The markers of oxidative stress and inflammation were studied in acute pancreatitis in transgenic rats exhibiting activated polyamine catabolism. In addition, the effect of bismethylspermine (Me2Spm) pretreatment, preventing pancreatitis in this model, on these mediators was investigated. Lipid peroxidation was increased at 6 and 24 h after induction of pancreatitis. These changes as well as the markedly decreased superoxide dismutase activity at 24 h were abolished by Me2Spm pretreatment. Glutathione level and catalase activity changed transiently, and the effect of Me2Spm was clear at 24 h. Serum inflammatory cytokine levels increased already at 4 h whereas NF-κB was distinctly activated only at 24 h. Me2Spm prevented the increase in TNF-α and IL-6 while it had no effect on NF-κB activation. These results show that typical inflammatory and, to a lesser degree, some oxidative stress mediators are involved and beneficially affected by the disease-ameliorating polyamine analogue in our pancreatitis model.

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 N1‐acetylation of spermidine or spermine by spermidine/spermine N1‐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 SSAT deficient 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 N1, N11‐diethylnorspermine (DENSPM) treatment compared to a significant reduction of the polymine 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.

Guide Molecule-driven Stereospecific Degradation of α-Methylpolyamines by Polyamine Oxidase

FAD-dependent polyamine oxidase (PAO; EC 1.5.3.11) is one of the key enzymes in the catabolism of polyamines spermidine and spermine. The natural substrates for the enzyme are N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine. Here we report that PAO, which normally metabolizes achiral substrates, oxidized (R)-isomer of 1-amino-8-acetamido-5-azanonane and N1-acetylspermidine as efficiently while (S)-1-amino-8-acetamido-5-azanonane was a much less preferred substrate. It has been shown that in the presence of certain aldehydes, the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermine and spermidine as substrates. Therefore, we examined the effect of several aldehydes on the ability of PAO to oxidize different enantiomers of α-methylated polyamines. PAO supplemented with benzaldehyde predominantly catalyzed the cleavage of (R)-isomer of α-methylspermidine, whereas in the presence of pyridoxal the (S)-α-methylspermidine was preferred. PAO displayed the same stereospecificity with both singly and doubly α-methylated spermine derivatives when supplemented with the same aldehydes. Structurally related ketones proved to be ineffective. This is the first time that the stereospecificity of FAD-dependent oxidase has been successfully regulated by changing the supplementary aldehyde. These findings might facilitate the chemical regulation of stereospecificity of the enzymes.

The activation of hepatic and muscle polyamine catabolism improves glucose homeostasis.

The mitochondrial biogenesis and energy expenditure regulator, PGC-1α, has been previously reported to be induced in the white adipose tissue (WAT) and liver of mice overexpressing spermidine/spermine N1-acetyltransferase (SSAT). The activation of PGC-1α in these mouse lines leads to increased number of mitochondria, improved glucose homeostasis, reduced WAT mass and elevated basal metabolic rate. The constant activation of polyamine catabolism produces a futile cycle that greatly reduces the ATP pools and induces 5′-AMP-activated protein kinase (AMPK), which in turn activates PGC-1α in WAT. In this study, we have investigated the effects of activated polyamine catabolism on the glucose and energy metabolisms when targeted to specific tissues. For that we used a mouse line overexpressing SSAT under the endogenous SSAT promoter, an inducible SSAT overexpressing mouse model using the metallothionein I promoter (MT-SSAT), and a mouse model with WAT-specific SSAT overexpression (aP2-SSAT). The results demonstrated that WAT-specific SSAT overexpression was sufficient to increase the number of mitochondria, reduce WAT mass and protect the mice from high-fat diet-induced obesity. However, the improvement in the glucose homeostasis is achieved only when polyamine catabolism is enhanced at the same time in the liver and skeletal muscle. Our results suggest that the tissue-specific targeting of activated polyamine catabolism may reveal new possibilities for the development of drugs boosting mitochondrial metabolism and eventually for treatment of obesity and type 2 diabetes.

Transgenic animals modelling polyamine metabolism-related diseases

Cloning of genes related to polyamine metabolism has enabled the generation of genetically modified mice and rats overproducing or devoid of proteins encoded by these genes. Our first transgenic mice overexpressing ODC (ornithine decarboxylase) were generated in 1991 and, thereafter, most genes involved in polyamine metabolism have been used for overproduction of the respective proteins, either ubiquitously or in a tissue-specific fashion in transgenic animals. Phenotypic characterization of these animals has revealed a multitude of changes, many of which could not have been predicted based on the previous knowledge of the polyamine requirements and functions. Animals that overexpress the genes encoding the inducible key enzymes of biosynthesis and catabolism, ODC and SSAT (spermidine/spermine N1-acetyltransferase) respectively, appear to possess the most pleiotropic phenotypes. Mice overexpressing ODC have particularly been used as cancer research models. Transgenic mice and rats with enhanced polyamine catabolism have revealed an association of rapidly depleted polyamine pools and accelerated metabolic cycle with development of acute pancreatitis and a fatless phenotype respectively. The latter phenotype with improved glucose tolerance and insulin sensitivity is useful in uncovering the mechanisms that lead to the opposite phenotype in humans, Type 2 diabetes. Disruption of the ODC or AdoMetDC [AdoMet (S-adenosylmethionine) decarboxylase] gene is not compatible with mouse embryogenesis, whereas mice with a disrupted SSAT gene are viable and show no harmful phenotypic changes, except insulin resistance at a late age. Ultimately, the mice with genetically altered polyamine metabolism can be used to develop targeted means to treat human disease conditions that they relevantly model.

α-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.

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.