We have generated a transgenic mouse line that overexpresses the rate-controlling enzyme of polyamine catabolism, spermidine/spermine N 1 -acetyltransferase. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N 1 -acetylspermidine, not normally found in mouse tissues, a massive accumulation of putrescine, and decreases in spermidine and/or spermine pools. The most striking phenotypic change was permanent hair loss at the age of 3 to 4 weeks which was typified histologically by the appearance of extensive follicular cysts in the dermis. The effect seemed attributable to putrescine interference with hair development, possibly with differentiation/proliferation of epidermal cells located in hair follicles. Female members of the transgenic line were found to be infertile apparently due to ovarian hypofunction and hypoplastic uteri. The findings demonstrate the utility of spermidine/spermine N 1 -acetyltransferase overexpression as an effective means for genetically modulating total tissue polyamine pools in transgenic animals and examining the developmental and oncogenic consequences.The well recognized association of polyamines with cell growth (1-3) is best illustrated by findings related to the key polyamine biosynthetic enzyme, ornithine decarboxylase (ODC).1 Although ODC is sharply but transiently increased by growth stimuli, it is constitutively activated during cell transformation induced by carcinogens, viruses, or oncogenes. Overexpression of ODC has been correlated with increased proliferative potential (4), tissue invasiveness (5), and in certain cell types, with oncogene-like transforming capabilities (6 -8). Thus far, ODC appears to be the only growth-related gene activated by the transcription factors c-myc (9 -11) and n-myc (12), suggesting a critical role for the enzyme in growth control. However, as indicated below, findings obtained in cell culture may not be directly applicable to conditions prevailing in vivo.To define the role of polyamines in proliferative processes associated with the whole animal, we have generated a number of transgenic mouse and rat lines that overexpress ODC and/or other polyamine biosynthetic enzymes. Given the importance of polyamine biosynthetic activity to cell growth, the phenotypic changes were unexpectedly mild. In transgenic mice overexpressing ODC, the most marked effect was inhibition of meiotic DNA synthesis during spermatogenesis (13) ultimately leading to male infertility (14). It is also noteworthy that lifelong overexpression of ODC in mouse tissues did not seem to increase the incidence of spontaneous tumors (15). The absence of more profound phenotypic changes in these mice may be attributable to the relatively minor changes observed in higher polyamine pools. Despite severalfold increases in ODC activity, polyamine pool disturbances were mainly confined to putrescine accumulation, and the pools of those polyamines considered to be more significantly involved in cell growth, spermidi...

The polyamines putrescine, spermidine and spermine are organic cations shown to participate in a bewildering number of cellular reactions, yet their exact functions in intermediary metabolism and specific interactions with cellular components remain largely elusive. Pharmacological interventions have demonstrated convincingly that a steady supply of these compounds is a prerequisite for cell proliferation to occur. The last decade has witnessed the appearance of a substantial number of studies, in which genetic engineering of polyamine metabolism in transgenic rodents has been employed to unravel their cellular functions. Transgenic activation of polyamine biosynthesis through an overexpression of their biosynthetic enzymes has assigned specific roles for these compounds in spermatogenesis, skin physiology, promotion of tumorigenesis and organ hypertrophy as well as neuronal protection. Transgenic activation of polyamine catabolism not only profoundly disturbs polyamine homeostasis in most tissues, but also creates a complex phenotype affecting skin, female fertility, fat depots, pancreatic integrity and regenerative growth. Transgenic expression of ornithine decarboxylase antizyme has suggested that this unique protein may act as a general tumor suppressor. Homozygous deficiency of the key biosynthetic enzymes of the polyamines, ornithine and S-adenosylmethionine decarboxylase, as achieved through targeted disruption of their genes, is not compatible with murine embryogenesis. Finally, the first reports of human diseases apparently caused by mutations or rearrangements of the genes involved in polyamine metabolism have appeared.Keywords: antizyme; ornithine decarboxylase; putrescine; spermidine/spermine N 1 -acetyltransferase; spermidine; spermine; transgenic mouse; transgenic rat. IntroductionThe cellular functions of the natural polyamines (putrescine, spermidine and spermine) are still largely unknown, although a vast number of studies have shown that these polycationic compounds are crucial to the growth and proliferation of mammalian cells. Pharmacological approaches are applied typically in studies aimed to unravel their functions in cellular metabolism and, admittedly, much valuable information has been generated with the use of specific inhibitors of polyamine biosynthesis. However, the last decade has produced a substantial number of experimental studies in which genetic engineering of polyamine metabolism has been used as a tool to elucidate their cellular functions. Studies with genetically engineered mice and rats have not only brought entirely new information about the involvement of polyamines in various physiological and pathophysiological processes but they have likewise challenged some of the conventional wisdoms. Mainly, four different approaches have been applied in the genetic engineering of experimental animals: (a) activation of polyamine biosynthesis through the overexpression of their biosynthetic enzymes; (b) activation of polyamine catabolism through the overexpression of the enzymes i...

Peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣) 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␣ 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␣ 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␣ 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.Type 2 diabetes is a growing epidemic worldwide. Defects in insulin secretion and insulin action are fundamental disorders of this disease (30). Several mechanisms regulating insulin secretion and insulin action have been identified, but none of them is likely to explain completely the risk of type 2 diabetes. Previous studies have revealed novel mechanisms, distinct from the insulin signaling pathway, for type 2 diabetes. Mootha et al. (36) identified a set of genes involved in oxidative phosphorylation (OXPHOS), the expression of which was coordinately decreased in human diabetic muscle. Similarly, Patti et al. (40) found the downregulation of OXPHOS not only in individuals with type 2 diabetes but also in their first-degree relatives. In both of these studies, decreased peroxisome proliferator-activated receptor (PPAR) ␥ coactivator 1␣ (PGC-1␣) expression was responsible for the downregulation of OX PHOS genes. In addition, the expression of PGC-1␣ has been shown to be downregulated in white adipose tissue (WAT) of insulin-resistant (15) and morbidly obese (50) subjects.PGC-1␣ was first identified as a coactivator of PPAR␥ (45), and it plays a critical role in the regulation of adaptive thermogenesis. Subsequent studies have demonstrated that PGC-1␣ regulates mitochondrial biogenesis (49), uncoupling (45, 56), fatty acid oxidation (61), OXPHOS (36), glucose transport in muscle (35), hepatic gluconeogenesis (64), and skeletal muscle fiber-type switching (44). PGC-1␣ is highly expressed in brown adipose tissue (BAT), heart, and skeletal muscle and moderately expressed in liver, but a low expression level is found in WAT. The expression of PGC-1␣ is ind...

BackgroundRapidly regenerating tissues need sufficient polyamine synthesis. Since the hair follicle (HF) is a highly proliferative mini-organ, polyamines may also be important for normal hair growth. However, the role of polyamines in human HF biology and their effect on HF epithelial stem cells in situ remains largely unknown.Methods and FindingsWe have studied the effects of the prototypic polyamine, spermidine (0.1–1 µM), on human scalp HFs and human HF epithelial stem cells in serum-free organ culture. Under these conditions, spermidine promoted hair shaft elongation and prolonged hair growth (anagen). Spermidine also upregulated expression of the epithelial stem cell-associated keratins K15 and K19, and dose-dependently modulated K15 promoter activity in situ and the colony forming efficiency, proliferation and K15 expression of isolated human K15-GFP+ cells in vitro. Inhibiting the rate-limiting enzyme of polyamine synthesis, ornithine decarboyxlase (ODC), downregulated intrafollicular K15 expression. In primary human epidermal keratinocytes, spermidine slightly promoted entry into the S/G2-M phases of the cell cycle. By microarray analysis of human HF mRNA extracts, spermidine upregulated several key target genes implicated e.g. in the control of cell adherence and migration (POP3), or endoplasmic reticulum and mitochondrial functions (SYVN1, NACA and SLC25A3). Excess spermidine may restrict further intrafollicular polyamine synthesis by inhibiting ODC gene and protein expression in the HF's companion layer in situ.ConclusionsThese physiologically and clinically relevant data provide the first direct evidence that spermidine is a potent stimulator of human hair growth and a previously unknown modulator of human epithelial stem cell biology.

We recently generated a transgenic mouse line with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase. Phenotypic changes in these animals included permanent loss of hair at the age of 3 wk. We have now further explored development of hair loss during early postnatal life. The first hair cycle appeared to be completed normally in the transgenic animals. At postnatal day 15, although macroscopically indistinguishable from their syngenic littermates, the transgenic animals already showed microscopically signs of hair follicle degeneration. Wild-type mice started their second anagen phase at day 27, whereas the transgenic animals did not display functional hair follicles at that time. Hair follicles were replaced by dermal cysts and epidermal utriculi. Analysis of skin polyamines revealed that the transgenic animals continuously overaccumulated putrescine. The view that an overaccumulation of putrescine was related to the disturbed hair follicle development was strengthened by the finding that doubly transgenic mice overexpressing, both spermidine/spermine N1-acetyltransferase and ornithine decarboxylase and with extremely high levels of putrescine in the skin, showed distinctly more severe skin changes compared with the singly transgenic animals. Interest ingly, in spite of their hairless phenotype, the spermidine/spermine N1-acetyltransferase transgenic mice, were significantly more resistant to the development of papillomas in response to the two-stage skin carcinogenesis. Analysis of skin polyamines indicated that the syngenic mice tripled their spermidine content when exposed to promotion, whereas the transgenic animals showed only modest changes. These results suggest that putrescine plays a pivotal part in normal hair follicle development.

Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.

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.

A large number of studies applying inhibitors of polyamine biosynthesis have indicated that these compounds are required for animal cell proliferation. Here we show, using a transgenic rat model with activated polyamine catabolism, that a certain critical concentration of the higher polyamines spermidine and spermine is required for liver regeneration. Partial hepatectomy of transgenic rats expressing spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of mouse metallothionein promoter strikingly induced the enzyme at 24 h and reduced hepatic spermidine by 80%. At that time, the weight of the liver remnant was significantly increased in syngenic rats and proliferating cell nuclear antigen (PCNA) labelling index was 20%, whereas the transgenic rats showed no liver weight gain and their PCNA-positive cells accounted for 0.5% of hepatocytes. Similarly, hepatic thymidine incorporation was markedly enhanced at this time point in syngenic, but not in transgenic, animals, whereas the rate of leucine incorporation was only marginally affected in the transgenic animals. At 3 days after operation, the spermidine pool in transgenic livers had increased to the pre-operative level, the remnant weight was significantly elevated and hepatic PCNA labelling index increased to 5%. N(1),N(11)-Diethylnorspermine, a powerful inducer of SSAT, inhibited liver weight gain and proliferative activity in both syngenic and transgenic rats. We found an extremely close correlation between hepatic spermidine, and less close between spermine, concentrations and PCNA labelling index during early liver regeneration. These results indicate that spermidine and/or spermine, but apparently not putrescine, are required for liver regeneration, yet at concentrations smaller than those normally found after partial hepatectomy.