Sequestered in the cytoplasm through phosphorylation dependent interactions with 14-3-3 proteins, TORCs (TORC1, TORC2, TORC3) shuttle to the nucleus following their dephosphorylation in response to cAMP and calcium signals, where they potentiate cellular gene expression by binding to CREB over relevant genes 8–12
. TORC1 contains conserved phosphorylation (Ser151) and ubiquitination (Lys575) sites that have been shown to modulate nuclear shuttling and protein stability in TORC2 13–15
(). By contrast with the ubiquitous pattern of TORC2 expression, however, TORC1 mRNA and protein are detected in the brain but not in other peripheral tissues such as liver, muscle, or adipose 16
(, sup. fig. 1
Figure 1 Mice with a knockout of TORC1 are obese and infertile. A. Top, schematic of TORC1 showing conserved CREB binding domain (CBD) as well as regulatory (REG) and transcriptional activation (TAD) domains; inset shows sequence of regulatory Ser151 site (bolded), (more ...)
Under basal conditions, TORC1 is highly phosphorylated in cultured hypothalamic GT1-7 cells () 17
. Exposure to cAMP or calcium activator triggers TORC1 dephosphorylation and nuclear translocation; phosphorylation-defective S151A mutant TORC1 is constitutively nuclear (sup. fig. 1
). Over-expression of wild-type TORC1 potentiates CRE-luciferase reporter activity in cells exposed to the cAMP activator forskolin (FSK) or calcium ionophore (A23187); co-treatment with FSK and A23187 increases reporter activity synergistically (). In line with its constitutive nuclear localization, phosphorylation-defective S151A TORC1 strongly upregulates CRE-luciferase activity even under basal conditions. The effects of TORC1 appear CREB dependent because co-expression of a dominant negative CREB polypeptide, called A-CREB 18
, disrupts reporter activity in cells exposed to FSK or A23187.
We evaluated the biological role of TORC1 in maintaining energy balance by insertional mutagenesis of the TORC1
gene with a promoter-less β-galactosidase (β-Geo) gene cassette (). Relative to control littermates, TORC1
mRNA and protein were undetectable in TORC1
−/− mice. Consistent with its regulation by the TORC1
promoter, CNS expression of the β-Geo cassette in TORC1
mutants mirrored that of endogenous TORC1 protein (, sup. fig. 2
). In addition to other brain regions, TORC1 expression was prominent in arcuate and ventromedial nuclei of the hypothalamus. Arguing against significant effects of TORC1 on brain development, however, Nissl-stained sections from TORC1
−/− brains appear comparable to wild-type (not shown).
TORC1 −/− mice were born at the expected Mendelian frequency, and they were indistinguishable from wild-type controls prior to weaning. Although their linear growth was unimpaired, adult TORC1 −/− mice were infertile; no offspring were obtained from either TORC1 −/− males or TORC1 −/− females mated with wild-type mice (0/6). Anatomically, TORC1 −/− female uteri appeared threadlike in appearance with noticeable thinning of the endometrium (). Although they had comparable numbers of mature follicles, TORC1 −/− ovaries contained no corpora lutea, markers of ovulation. Indeed, circulating concentrations of pituitary luteinizing hormone (LH), a key regulator of ovulation, were down-regulated in TORC1 mutants compared to controls ().
In parallel with these reproductive defects, male and female TORC1
−/−mice also developed persistent obesity beginning at 9 weeks of age on a normal chow diet (); TORC1
+/− heterozygotes had intermediate weights relative to wild-type and TORC1
−/− homozygotes. White adipose mass was increased 2–3 fold in TORC1
mutant mice, whereas other tissues were relatively unaffected (: sup. fig. 3
). Taken together, these results indicate that the effects of TORC1 on body weight are specific to white adipose, affect both males and females, and vary with gene dosage.
We performed metabolic studies to determine why TORC1 mutant mice gain more weight. Compared with wild-type littermates, TORC1 −/− animals ate more and they expended less energy at 12 to 14 weeks of age, as determined by physical activity and oxygen consumption monitoring (). Consistent with this disruption, TORC1 −/− mice were hyperglycemic and hypertryglyceridemic at 9 months of age (). Pointing to the development of insulin resistance, circulating levels of insulin were increased in TORC1 +/− mice and to a greater extent in TORC1 −/− homozygotes; they were glucose intolerant by IP glucose-tolerance testing (). In line with their obesity, circulating leptin concentrations were also upregulated in TORC1 −/− mice ().
Figure 2 TORC1 −/− mice are hyperphagic and have reduced energy expenditure. A. Left, cumulative food intake over a 45 day interval, beginning at 12 weeks of age, in TORC1 −/−, TORC1 +/−, and wild-type littermates maintained (more ...)
During feeding, increases in circulating concentrations of leptin as well as insulin and glucose promote satiety and fertility, in part through the activation of arcuate neurons in the hypothalamus 4,19–21
. Realizing that TORC1
−/− mice are hyperphagic, obese, and infertile, we wondered whether TORC1 is required for the activation of relevant hypothalamic programs in response to feeding signals. Although chronic leptin infusion substantially reduced food intake and body weight in control animals, it had minimal effects on TORC1
−/− mice () 22
. Arguing against potential effects on leptin bioavailability, chronic leptin infusion promoted STAT3 phosphorylation a comparable extent in arcuate neurons of wild-type and TORC1
−/− animals ().
Consistent with the ability for leptin to increase hypothalamic STAT3 activity, mRNA amounts for proopiomelanocortin (POMC
), neuropeptide Y (NPY
), and Agouti Related Peptide (AgRP
), regulatory targets of the LRb-STAT3 pathway that encode anorexigenic (POMC
) and orexigenic (NPY, AgRP
) neuropeptides, were comparable between TORC1
mutants and controls (sup. fig. 4
). Indeed, signaling through the downstream melanocortin pathway also appear normal in TORC1
mutants, because intra-peritoneal (IP) administration of the alpha melanocyte stimulating hormone (α-MSH) analog MTII 23
inhibited food intake to the same extent in both wild-type and TORC1
−/− mice (sup fig. 5
We used leptin deficient ob/ob
mice to determine whether TORC1 activity is disrupted in obesity. Supporting this idea, ob/ob
mice had increased amounts of phosphorylated, inactive TORC1 in the hypothalamus (). IP leptin injection increased amounts of dephosphorylated, nuclear TORC1 protein in arcuate cells of ob/ob
mice (). Consistent with a parallel role for nutrient signaling, IP glucose administration also promoted the accumulation of dephosphorylated TORC1 in the hypothalamus (sup fig. 6
). Correspondingly, TORC1 was nuclear-localized in arcuate cells during ad libitum feeding but remained cytoplasmic in other regions of the CNS (sup fig. 6
). Taken together, these results indicate that hormone and nutrient signals modulate hypothalamic TORC1 activity under lean conditions, and that TORC1 activity is disrupted in obesity.
We performed gene profiling studies to identify hypothalamic genes that contribute to the metabolic and reproductive phenotypes of TORC1
mutant mice. This analysis revealed that mRNAs for the neuropeptide genes Cocaine and Amphetamine Regulated Transcript (CART
) and KISS1
were down-regulated in TORC1
−/− animals. CART and KISS1 have been found to mediate effects of LRb signaling on feeding and fertility 6,7,24–27
. Indeed, CART is co-expressed with POMC in arcuate neurons, where it inhibits food intake in response to leptin 27
, while KISS1 expression in the arcuate promotes reproductive function by stimulating the secretion of hypothalamic gonadotropin releasing hormone (GnRH) 28,29
. Similar to TORC1
−/− animals, mice with a knockout of KISS1
have low circulating concentrations of LH, exhibit abnormal uterine morphology, and are infertile 30
. We confirmed that CART
genes are down-regulated in TORC1
−/− mice by Q-PCR and in situ hybridization analysis (). As well, hypothalamic staining for kisspeptin, a cleavage product of the KISS1 precursor, was dramatically reduced in arcuate neurons of TORC1
−/− mice (). Importantly, TORC1 driven β-gal mRNA was co-expressed with CART and KISS1 neuropeptides in arcuate cells by dual immunohistochemistry and in situ
Figure 3 Reduced hypothalamic expression of anorexigenic and reproductive neuropeptide genes in TORC1 −/− mice. A. Q-PCR (left) (*; P<0.05, n=3) and in situ hybridization (right) analysis of CART mRNA amounts in wild-type and TORC1 −/− (more ...)
Realizing that CART
promoters contain CREB binding sites (TGACG/CGTCA) that are conserved between mouse, rat, and human homologs, we considered that TORC1 may regulate both genes via a direct mechanism. Supporting this idea, CREB has been shown to promote CART
gene expression in response to cAMP 31–33
, although a similar role for KISS1
regulation has not been established. In keeping with its effects on TORC1 dephosphorylation, A23187 treatment increased endogenous mRNA amounts for CART
in GT1-7 cells; this induction was blocked in cells depleted of TORC1 by RNAi-mediated knockdown (). Exposure to A23187 or FSK also increased CART
reporter activities in transient assays (); over-expression of wild-type TORC1, and to a greater extent phosphorylation-defective (S151A) TORC1, enhanced transcription from both promoters. Consistent with the role of CREB in promoting TORC1 recruitment, expression of dominant negative ACREB inhibitor blocked induction of CART
reporters by FSK and A23187 ().
Figure 4 CART and KISS1 genes are direct targets of TORC1 and CREB in the hypothalamus. A. and B. Transient transfection assay of HEK293T cells using CART-luciferase (A) and KISS1-luciferase (B) reporters. Exposure to FSK (1μM) and A23187 (1μM), (more ...)
We performed chromatin immunoprecipitation assays (ChIPs) to determine whether TORC1 and CREB regulate CART and KISS1 genes directly. In line with its constitutive nuclear localization, CREB occupied CART and KISS1 genes in GT1-7 cells comparably under basal conditions and following exposure to FSK or A23187 (). TORC1 occupancy over the CART and KISS1 genes was low under basal conditions - when TORC1 is sequestered in the cytoplasm -and increased following exposure to cAMP or calcium activator - when dephosphorylated TORC1 shuttles to the nucleus and binds to CREB. Consistent with its effect on amounts of nuclear TORC1 protein in the hypothalamus, leptin administration IP also increased TORC1 recruitment to CART and KISS1 promoters in ob/ob mice, while CREB occupancy over both genes was constitutive (). Taken together, these results indicate that CREB and TORC1 regulate hypothalamic CART and KISS1 gene expression through a direct mechanism.
Based on their importance for transcriptional induction in response to cAMP and calcium, we wondered whether TORC1 and CREB are also required for effects of leptin on neuropeptide gene expression. Exposure to leptin increased CART and KISS1 reporter activities synergistically with FSK in cells co-transfected with a leptin receptor (LRb) expression vector; these effects were augmented by over-expression of TORC1 (). Similar to its effects on cAMP and calcium signaling, ACREB inhibitor blocked induction of both promoters in cells treated with leptin.
Our results indicate that TORC1 is activated by hormonal and nutrient signals in the hypothalamus, where it promotes energy balance and fertility by enhancing CREB activity over relevant neuropeptide genes. Similar to leptin deficent ob/ob
−/− females have abnormal uterine morphology and low circulating LH levels 34,35
. By contrast with ob/ob
animals, however, TORC1
mutant mice are only moderately obese, potentially reflecting compensatory effects of other TORC family members. Consistent with this idea, TORC2 is also expressed in the hypothalamus where it undergoes nuclear shuttling in response to feeding stimuli 36
In addition to its effects on JAK2/STAT3 signaling, leptin has also been reported to modulate cation channel activity 37,38
and to inhibit the activity of the energy sensing Ser/Thr kinase AMPK 21
. Based on the ability for calcium and AMPK pathways to regulate TORC1 activity, we imagine that these pathways may also mediate effects of leptin on TORC1 in the hypothalamus.
The importance of TORC1 in energy balance appears to be evolutionarily conserved; Drosophila
TORC, the single fly homolog of mammalian TORCs, is also expressed primarily in the brain where it regulates energy consumption as well as glucose and lipid homeostasis 39
TORC and mammalian TORC1 are regulated through phosphorylation by Salt Inducible Kinases (SIKs) and other members of the AMPK family 10,15,39
. Indeed, knockdown of Drosophila SIK2
in neurons promotes starvation resistance and improves energy balance, suggesting that this Ser/Thr kinase also contributes to effects of hormonal and nutrient signals on hypothalamic TORC1 activity.
Obesity risk in humans has a strong genetic component, which is thought to involve heterozygous loss-of-function mutations in genes that, individually, may display only modest phenotypic changes 40,41
. The presence of hyperphagia, increased adiposity, and insulin resistance even in heterozygous TORC1
+/− mice suggests that mutations in the TORC1
gene may also promote the development of obesity in humans. Future epidemiological studies of TORC1 gene mutations in affected populations should provide further insight in this regard.