Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Here, we show that tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. We show that Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. We propose that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. Our studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors.

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic ␤-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on ␤-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic ␤ cells (␤TSC2 ؊/؊ mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual ␤ cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the ␤TSC2 ؊/؊ mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of ␤ cells. These results thus indicate that TSC2 regulates pancreatic ␤-cell mass in a biphasic manner.

Impairment of reciprocal social interaction is a core symptom of autism spectrum disorder. Genetic disorders frequently accompany autism spectrum disorder, such as tuberous sclerosis complex caused by haploinsufficiency of the TSC1 and TSC2 genes. Accumulating evidence implicates a relationship between autism spectrum disorder and signal transduction that involves tuberous sclerosis complex 1, tuberous sclerosis complex 2 and mammalian target of rapamycin. Here we show behavioural abnormalities relevant to autism spectrum disorder and their recovery by the mammalian target of rapamycin inhibitor rapamycin in mouse models of tuberous sclerosis complex. In Tsc2+/− mice, we find enhanced transcription of multiple genes involved in mammalian target of rapamycin signalling, which is dependent on activated mammalian target of rapamycin signalling with a minimal influence of Akt. The findings indicate a crucial role of mammalian target of rapamycin signalling in deficient social behaviour in mouse models of tuberous sclerosis complex, supporting the notion that mammalian target of rapamycin inhibitors may be useful for the pharmacological treatment of autism spectrum disorder associated with tuberous sclerosis complex and other conditions that result from dysregulated mammalian target of rapamycin signalling.

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1 ؉/؊ ) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1 ؉/؊ mice was apparently slower than that in Tsc2 ؉/؊ mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.T uberous sclerosis (TS) is an autosomal dominantly inherited disease characterized by the development of hamartomas and benign tumors in various organs such as brain, kidney, and heart (1). A germ-line mutation in either TSC1 or TSC2 genes (2, 3), both of which act as tumor suppressors (4, 5), is a genetic factor responsible for pathogenesis of TS. The similar symptoms of TS patients associated with TSC1 or TSC2 mutations suggest that the products of TSC1 and TSC2 are involved in a common physiological pathway (1, 6). TSC1 encodes a protein with a molecular mass of Ϸ130 kDa, hamartin, which contains a coiled-coil domain in the carboxyl-terminal half (3). TSC2 encodes tuberin, a rap1-GTPase activating protein homology domain-containing protein with a molecular mass of Ϸ180 kDa (2). Although several studies concerned with functions of these products have been reported, in vivo functions of them remain to be elucidated (1,7,8).The tumor suppressor function of TSC2 became evident by studies of rodents with a germ-line Tsc2 mutation such as the Eker rat (9-13) and Tsc2 knockout mice (14, 15). Both heterozygous Tsc2 mutant rats and mice develop hereditary renal tumors and extra-renal tumors carrying a second hit of Tsc2 gene (14-17). Homozygosity of Tsc2 mutation leads to the embryonic lethality both in rats (9, 18) and mice (15,16), indicating that the function of tuberin is essential for mammalian development.We also isolated a rat homologue of TSC1 (Tsc1) and analyzed its mutation in chemically induced renal tumors in wild-type rats, in which Tsc2 mutations were found with high frequency (Ϸ50%) (19). In those tumors, we found Tsc1 mutations in a case with no Tsc2 mutation (19). These results suggest that mutations of Tsc1 and Tsc2 are involved in the development of chemically induced renal tumors in rats, although the latter is more common. These systems o...