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...

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.

The polyamines are growth factors in both normal and cancer cells. As the intracellular polyamine content correlates positively with the growth potential of that cell, the idea that depletion of polyamine content will result in inhibition of cell growth and, particularly tumour cell growth, has been developed over the last 15 years. The polyamine pathway is therefore a target for development of rationally designed, antiproliferative agents. Following the lessons from the single enzyme inhibitors (alpha-difluoromethylornithine DFMO), three generations of polyamine analogues have been synthesised and tested in vitro and in vivo. The analogues are multi-site inhibitors affecting multiple reactions in the pathway and thus prevent the up-regulation of compensatory reactions that have been the downfall of DFMO in anticancer chemotherapy. Although the initial concept was that the analogues may provide novel anticancer drugs, it now seems likely that the analogues will have wider applications in diseases involving hyperplasia.

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N 1 -acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N 1 ,N 11 -diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C 14 ]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N 1 -acetylspermidine and putrescine were readily detectable in N 1 ,N 11 -diethylnorspermine-exposed wild-type cells but not in SSATdeficient cells. Similar experiments with [C 14 ]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.The oxidative catabolism of the higher polyamines spermidine and spermine is accomplished by the concerted action of two different enzymes, namely spermidine/spermine N 1 -acetyltransferase (SSAT) 1 and polyamine oxidase (PAO). Cytosolic SSAT N 1 acetylates both spermidine and spermine whereafter they serve as substrates for peroxisomal PAO (1). As PAO strongly prefers acetylated polyamines to the unmodified polyamines as its substrates, SSAT is generally considered as the rate-controlling enzyme in the back-conversion of spermidine and spermine (2). The final product of the catabolism of spermidine is putrescine whereas the oxidation of spermine or N 1 -acetylspermine yields spermidine. In addition to the polyamines, PAO action also generates acetamido propanal and hydrogen peroxide.During recent years considerable attention has been paid to SSAT as a target for cancer chemotherapy. A number of compounds, among them natural polyamines and their alkylated derivatives, strikingly induce SSAT and subsequently deplete cellular polyamine pools resulting in overt cytotoxicity (2). A large number of studies suggest that depletion of polyamines and growth inhibition by polyamine analogues are closely related to the extent of SSA...