Physiology homework help

hemichannel makes it a major contributor to

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ionic dysregulation in ischemia. Second, Px1

hemichannel opening may result in efflux of

glucose and adenosine triphosphate (ATP),

further compromising the neuron_s recovery
from an ischemic insult. Consistent with this

was our observation that fluorescent dyes

became membrane-permeable only during

OGD. Hemichannels are putative conduits for

ATP release from astrocytes (21) and in the

cochlea (22). Third, the large amplitude of

the Px1 hemichannel current at holding po-

tentials near the neuron_s resting membrane
potential (È –60 mV) indicates that these

currents likely contribute substantially to

Banoxic depolarization,[ a poorly understood
but well-recognized and key component of

ischemic neuronal death (2, 23, 24). There-

fore, hemichannel opening may be an impor-

tant new pharmacological target to prevent

neuronal death in stroke.

References and Notes
1. A. J. Hansen, Physiol. Rev. 65, 101 (1985).
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3. M. Kamermans et al., Science 292, 1178 (2001).
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(2002).
5. R. P. Kondo, S. Y. Wang, S. A. John, J. N. Weiss,

J. I. Goldhaber, J. Mol. Cell. Cardiol. 32, 1859 (2000).
6. H. Li et al., J. Cell Biol. 134, 1019 (1996).
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(2004).
8. R. Bruzzone, M. T. Barbe, N. J. Jakob, H. Monyer,

J. Neurochem. 92, 1033 (2005).
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H. Monyer, Proc. Natl. Acad. Sci. U.S.A. 100, 13644
(2003).

10. See supporting material on Science Online.
11. J. Gao et al., Neuron 48, 635 (2005).
12. M. Aarts et al., Cell 115, 863 (2003).
13. C. Tomasetto, M. J. Neveu, J. Daley, P. K. Horan, R. Sager,

J. Cell Biol. 122, 157 (1993).
14. G. Feng et al., Neuron 28, 41 (2000).
15. A. Nimmerjahn, F. Kirchhoff, J. N. Kerr, F. Helmchen,

Nat. Methods 1, 31 (2004).
16. G. Sohl, S. Maxeiner, K. Willecke, Nat. Rev. Neurosci. 6,

191 (2005).

17. J. C. Saez, M. A. Retamal, D. Basilio, F. F. Bukauskas,
M. V. Bennett, Biochim. Biophys. Acta 1711, 215 (2005).

18. R. J. Thompson, M. H. Nordeen, K. E. Howell,
J. H. Caldwell, Biophys. J. 83, 278 (2002).

19. M. L. Fung, G. G. Haddad, Brain Res. 762, 97 (1997).
20. H. Benveniste, J. Drejer, A. Schousboe, N. H. Diemer,

J. Neurochem. 43, 1369 (1984).
21. C. E. Stout, J. L. Costantin, C. C. Naus, A. C. Charles,

J. Biol. Chem. 277, 10482 (2002).
22. H. B. Zhao, N. Yu, C. R. Fleming, Proc. Natl. Acad. Sci.

U.S.A. 102, 18724 (2005).
23. T. R. Anderson, C. R. Jarvis, A. J. Biedermann, C. Molnar,

R. D. Andrew, J. Neurophysiol. 93, 963 (2005).
24. G. G. Somjen, Physiol. Rev. 81, 1065 (2001).
25. Supported by the Canadian Institutes for Health Research

and the Canadian Stroke Network. B.A.M. has a Tier 1
Canada Research Chair in Neuroscience and a Michael
Smith Foundation for Health Research distinguished
scholar award. We thank Y.-T. Wang, C. C. Naus, and
T. Snutch for critical reading of the manuscript.

Supporting Online Material
www.sciencemag.org/cgi/content/full/312/5775/924/DC1
Materials and Methods

14 February 2006; accepted 31 March 2006
10.1126/science.1126241

Hypothalamic mTOR Signaling
Regulates Food Intake
Daniela Cota,1 Karine Proulx,1 Kathi A. Blake Smith,1 Sara C. Kozma,2 George Thomas,2

Stephen C. Woods,1 Randy J. Seeley1*

The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates
cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR
signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating
energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the
hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate
nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases
food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the
inhibition of mTOR signaling blunts leptin’s anorectic effect. Thus, mTOR is a cellular fuel sensor
whose hypothalamic activity is directly tied to the regulation of energy intake.

A
subset of neurons in the central nervous

system (CNS) plays a role in regulating

both blood plasma fuel levels and nu-

trient intake (1, 2). An emerging concept is that

specific neuronal populations integrate fuel avail-

ability signals with signals mediated by hor-

mones such as leptin (3). However, the signaling

pathways that are involved are poorly understood.

In peripheral cells, the mammalian mTOR

signaling pathway integrates nutrient signals

with hormonal signals to control growth and

development (4, 5). mTOR is a highly con-

served serine-threonine kinase, which, in the

presence of mitogens and available nutrients

(including amino acids), stimulates protein

synthesis and inhibits autophagy (6). In vitro,

cellular levels of adenosine triphosphate (ATP)

increase mTOR signaling, and mTOR itself is

thought to serve as an ATP sensor (7). mTOR

thus functions as a checkpoint by which cells

sense and decode changes in energy status,

which in turn determines the rate of cell growth

and proliferation (6). Complete loss of TOR

function is lethal in mice (8); in Drosophila,

defects in TOR signaling result in the for-

mation of smaller cells in all tissues (9).

Conversely, increased or otherwise aberrant

mTOR activity has been linked to the de-

velopment of cancer, diabetes, and obesity

(10, 11). As is consistent with the develop-

ment of these diseases, the activation of the

mTOR pathway is markedly elevated in the

liver and in the skeletal muscle of insulin-

resistant obese rats maintained on a high-fat

diet (12), whereas the absence of the down-

stream mTOR target ES6 kinase 1 (S6K1)^
protects against diet-induced obesity and en-

hances insulin sensitivity in mice (13). Given

these observations, we hypothesized that

mTOR might integrate cellular fuel status

with hormonal-related signaling in specific

populations of neurons that use this informa-

tion to regulate food intake.

To test this hypothesis, we used antibodies to

localize mTOR and two downstream targets of

mTOR action ES6K1 and S6 ribosomal protein
(S6) (5, 6)^ in the rat brain. Consistent with
previous work (14), antibodies recognizing total

mTOR had a ubiquitous distribution in the

CNS, and there was scattered expression of

specific phosphorylation of mTOR at Ser2448

(pmTOR) in extra-hypothalamic areas, in-

cluding the hippocampus, thalamus, and cor-

tex. In the hypothalamus, pmTOR was highly

localized in the paraventricular (PVN) and

arcuate (ARC) nuclei (fig. S1A) (15). Like-

wise, total S6K1 stained broadly throughout

the CNS, whereas the hypothalamic expres-

sion of the activated form of S6K1, phos-

phorylated at Thr389 (pS6K1), was also

largely limited to the PVN and ARC. Further,

dual labeling for pmTOR and pS6K1 revealed

that they are localized in the same cells in

both of these regions (fig. S1B). Although

most of these cells appear to be neurons, some

may be glia.

The ARC contains at least two populations of

neurons that are linked to the regulation of en-

ergy balance and whose activity is regulated by

leptin: (i) orexigenic neurons that express both

neuropeptide Y (NPY) and agouti-related pep-

tide (AgRP) and (ii) anorexigenic neurons that

express proopiomelanocortin (POMC) and

cocaine- and amphetamine-regulated transcript

(CART). Both pmTOR and pS6K1 were found

in È90% of ARC NPY/AgRP neurons (Fig.

1A), whereas only 45% of ARC POMC/CART

neurons revealed phosphorylation of these

proteins (Fig. 1B).

We next investigated whether changes in the

body_s energy status modulate mTOR signaling

1
Department of Psychiatry,

2
Department of Genome Sci-

ence, University of Cincinnati, Genome Research Institute,
2170 East Galbraith Road, Cincinnati, OH 45237, USA.

*To whom correspondence should be addressed. E-mail:
Randy.Seeley@uc.edu

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www.sciencemag.org SCIENCE VOL 312 12 MAY 2006 927

in the brain. There was a notable decrease in

both hypothalamic pS6K1 and S6 phosphoryl-

ated at Ser240 and Ser244 (pS6) in rats that were

fasted for 48 hours as compared with rats that

were re-fed for 3 hours (Fig. 2A), but no

significant changes in protein phosphorylation

were found in extra-hypothalamic areas, such

as the cortex and hippocampus (fig. S2A). After

a 48-hour fast, the number of hypothalamic

cells expressing pmTOR and pS6K1 was sig-

nificantly decreased in the ARC, whereas no

significant changes were observed in the PVN

(Fig. 2B and fig. S2B). Thus, mTOR activity in

the ARC is low when available fuels are low

and the organism is predisposed to consume

more calories.

If mTOR signaling is linked to the regu-

lation of energy balance in the CNS, then

manipulations of mTOR activity in the hypo-

thalamus would be predicted to alter food

intake. In a variety of model systems, mTOR

activity is sensitive to levels of branched-chain

amino acids, especially L-leucine (16, 17). If

increased hypothalamic mTOR signaling sup-

presses food intake, then the administration of

L-leucine in the vicinity of the ARC (15) should

produce anorexia. Intracerebroventricular ad-

ministration of L-leucine E1.1 mg in 2 ml of
phosphate-buffered saline (PBS) into the third

ventricle^ to 24-hour fasted rats before the
onset of the dark cycle caused a decrease in

food intake that was apparent 4 hours after

treatment and lasted for 24 hours (Fig. 3A). The

L-leucine–induced anorexia was accompanied

by significant weight loss (Fig. 3B). In separate

experiments, L-leucine also decreased food

intake during the light cycle (fig. S3, A and B).

Unlike L-leucine, the intracerebroventricular

administration of L-valine, another branched-

chain amino acid, did not potently stimulate

mTOR signaling (16, 17), nor did it affect food

intake and body weight (fig. S3, C and D). If

our hypothesis is correct, doses of L-leucine

that suppress food intake should also increase

mTOR activity in the hypothalamus. Consistent

with this, 45 min after intracerebroventric-

ular administration of L-leucine, levels of

pS6K1 were significantly increased in the

hypothalamus (fig. S3E) whereas no changes

were observed in pS6 levels at that time

point (pS6/S6 L-leucine, 94.8 T 5.3% versus
pS6/S6 PBS, 100 T 3.3%, P 0 0.4).
L -leucine–induced anorexia was also accom-

panied by significantly reduced NPY mRNA

levels in the ARC (NPY mRNA after L-leucine,

85.6 T 4.3% versus NPY mRNA after PBS,
100 T 5.0%, P G 0.05), suggesting that hy-
pothalamic mTOR signaling is selectively

linked to the NPY system. Moreover, control

experiments revealed that the L-leucine–

induced anorexia was not due to the devel-

opment of conditioned taste aversions (fig.

S3, F and G).

We next investigated the effects of the

well-characterized mTOR inhibitor rapamycin

(18). Intracerebroventricular administration of

Fig. 1. Localization of
mTOR signaling in the
rat hypothalamus. (A)
Immunohistochemistry
showing colocalization
(yellow, bottom left) of
pS6K1 (green, top left)
and AgRP (red, top
right) in the ARC of the
hypothalamus. Scale
bar, 50 mm. (Bottom
right) High-resolution
image depicting the
colocalization of pS6K1
and AgRP (arrows). 3rd
indicates the third ven-
tricle. Scale bar, 10 mm.
(B) Immunohistochem-
istry showing colocal-
ization (yellow, bottom
left) of pS6K1 (green,

top left) and POMC (red, top right) in the ARC of the hypothalamus. Scale bar, 50 mm. (Bottom right) High-resolution image depicting the colocalization of
pS6K1 and POMC (arrows). The asterisk indicates a cell expressing only pS6K1. Scale bar, 10 mm.

Fig. 2. Modulation of
hypothalamic mTOR sig-
naling by energy status.
(A) (Left) Representative
Western blot from re-
fed rats or rats fasted
for 48 hours. b-actin
was the loading control.
(Right) Quantification
by image analysis of
hypothalamic S6K1
and S6 phosphoryl-
ation. Error bars indi-
cate SEM. *P G 0.05
versus rats in the re-fed condition. Five brains were examined for each
condition. (B) Quantification of immunoreactivity for pmTOR in the ARC
and PVN of rats fed ad libitum and rats fasted for 48 hours. The mean T

SEM of the number of cells positive for pmTOR is expressed as a percentage
of mTOR-labeled cells. *P G 0.05 versus rats in the fed–ad libitum condi-
tion. Five brains were examined for each condition.

REPORTS

12 MAY 2006 VOL 312 SCIENCE www.sciencemag.org928

rapamycin E50 mg in 2 ml of dimethyl sulfoxide
(DMSO)^ rapidly inhibited hypothalamic S6K1
and S6 phosphorylation (Fig. 3C) and signifi-

cantly increased the short-term intake of chow

in pre-satiated rats, which were exposed to a

highly palatable diet (Ensure) during the light

cycle (fig. S4).

To determine whether the stimulation of

hypothalamic mTOR signaling is required for

the L-leucine–induced reduction of food intake,

we combined intracerebroventricular adminis-

tration of rapamycin, at a dose that does not

affect food intake, with a subsequent intracere-

broventricular injection of L-leucine. Rapamycin

significantly inhibited the L-leucine–induced

anorexia 4 hours after the administration of

the amino acid (Fig. 3D), an effect that per-

sisted up to 24 hours after treatment (Fig.

3D). Moreover, whereas L-leucine–treated rats

lost a significant amount of weight, the rapa-

mycin pre-treatment was associated with

changes in body weight that were comparable

to those observed in vehicle-treated animals

(Fig. 3E).

A number of hormones and cytokines medi-

ate their cellular effects through the mTOR

signaling pathway. For example, the activation

of mTOR and S6K1 by insulin is dependent on

the phosphatidylinositol 3-kinase (PI3K)/Akt

pathway (10) and in the hypothalamus, the ano-

rectic actions of both insulin and leptin can be

blocked by the inhibition of PI3K (19, 20). To

determine whether leptin_s anorectic effects
depend on mTOR activation, we examined hy-

pothalamuses from leptin-treated rats E10 mg
in 2 ml of saline, intracerebroventriculary (icv)^
2 hours after the administration of the hor-

mone (Fig. 4A). Leptin treatment increased

the phosphorylation of both S6K1 and S6 rel-

ative to saline. Moreover, a significant positive

correlation was found between the hypotha-

lamic phosphorylation levels of signal trans-

ducer and activator of transcription 3 (pSTAT3)

and both pS6K1 (Pearson_s r 0 0.6, P 0 0.03)
and pS6 (Pearson_s r 0 0.86, P 0 0.0006). To
determine whether increased mTOR activity

is required for the leptin-induced anorexia,

we combined the administration of leptin

with rapamycin. Rapamycin greatly attenu-

ated the anorexia and body weight loss that

was induced by leptin over a 24-hour period

(Fig. 4, B and C). This contrasts with the re-

sults obtained with the potent melanocortin

receptor 3 and 4 agonist, melanotan II (MTII)

(0.26 mg in 1 ml of saline, icv), whose effect on
food intake and body weight was unaffected

by rapamycin (fig. S5, A and B). This find-

ing suggests that the interaction between

mTOR signaling and leptin is relatively

specific.

Our data highlight an important role for hy-

pothalamic mTOR signaling in food intake

and energy balance regulation in a mammalian

model. Analogous changes in S6K activity af-

fect the feeding behavior of Drosophila larvae

Fig. 3. L-leucine and rapamycin
oppositely modulate hypothalamic
mTOR signaling. (A and B) Intra-
cerebroventricular administration
of L-leucine decreases food intake
(A) and body weight gain (B) in rats
fasted for 24 hours. The mean T
SEM of 6 to 7 rats used for each
treatment group is shown. *P G
0.05 versus PBS-treated rats; #P G
0.05 versus rats treated with 0.2 mg
of L-leucine in 2 ml of PBS (leu 0.2).
Leu 1.1, treatment with 1.1 mg of
L-leucine in 2 ml of PBS. (C) Rapa-
mycin (50 mg in 2 ml of DMSO, icv)
inhibits hypothalamic mTOR sig-
naling. (Left) Representative West-
ern blot from DMSO- or rapamycin
(rapa)–treated rats. b-actin was
the loading control. (Right) Quan-
tification by image analysis of
hypothalamic S6K1 and S6 phos-
phorylation. Error bars indicate
SEM. *P G 0.05 versus DMSO-
treated rats. Three brains were
examined for each condition. (D
and E) Rapamycin (25 mg in 2 ml
of DMSO, icv) blocks the L-leucine–
induced effects on food intake (D)
and body weight (E). The mean T
SEM of 6 to 7 rats used for each
treatment group is shown. *P G
0.05 versus DMSO/PBS-treated
rats; #P G 0.05 versus rapamycin/
leucine-treated rats.

Fig. 4. Role of hypothalamic mTOR
signaling in the central anorectic
action of leptin. (A) Leptin (10 mg
in 2 ml of saline, icv) increases
hypothalamic mTOR signaling.
(Left) Representative Western blot
from saline- or leptin-treated rats.
b-actin was the loading control.
(Right) Quantification by image
analysis of hypothalamic S6K1
and S6 phosphorylation. Error bars
indicate SEM. *P G 0.05 versus
saline-treated rats. Five brains were

examined for each condition. (B and C) Rapamycin (25 mg in 2 ml of DMSO, icv) blocks the central action
of leptin (10 mg in 2 ml of saline, icv) on food intake (B) and body weight changes (C). The data are shown
as the mean T SEM. Saline- or DMSO-treated rats (n 0 3) were compared with treated rats (n 0 8). *P G
0.05 versus saline/DMSO- or saline/rapamycin-treated rats; #P G 0.05 versus leptin/rapamycin-treated rats.
(D) Proposed model for the role of mTOR signaling in the hypothalamic regulation of energy balance.
Anorectic signals, such as amino acids (L-leucine), leptin, and re-feeding, increase hypothalamic mTOR
signaling. Increased mTOR activity leads to a decrease in food intake. Rapamycin inhibits hypothalamic
mTOR, causing an increase in food intake. Opposite to their effect on mTOR, leptin and re-feeding decrease
hypothalamic AMPK (27). mTOR is inhibited by AMPK-dependent mechanisms in vitro (28). Thus,
reciprocal interaction might exist between hypothalamic mTOR and AMPK.

REPORTS

www.sciencemag.org SCIENCE VOL 312 12 MAY 2006 929

(21). Cells suppress protein synthesis when

there is insufficient energy or amino acid

substrate, and mTOR plays a critical role in

this regulatory mechanism. Similarly, the

CNS must monitor fuel and substrate levels

to coordinate the availability of fuel for the

entire organism. Whereas a signal of low fuel

in a single peripheral cell may curtail its own

protein synthesis, causing a localized cata-

bolic action, a signal of low fuel in key areas

of the CNS might be expected to increase

food intake, producing an overall anabolic

effect.

CNS circuits can directly sense glucose

and specific fatty acids, integrating this in-

formation to modulate caloric intake (1, 22).

Our findings expand the knowledge of these

CNS sensing mechanisms to include a pro-

tein component: the amino acid L-leucine.

However, the degree to which amino acids

act as physiological signals to centrally mod-

ulate energy balance, in situations other than

amino acid imbalance (23), is unclear. The

ability of L-leucine to activate mTOR in the

hypothalamus and to inhibit food intake may

be an example of CNS circuits using an evo-

lutionarily conserved signaling mechanism

as a fuel sensor rather than as an amino acid

sensor.

An important feature of the mTOR pathway

is that its activity is modulated by growth factors

and hormones. Our data indicate that hypo-

thalamic mTOR signaling can be modulated by

leptin and that leptin_s effect on food intake is
mTOR-dependent.

One implication of our experiments relates to

recent findings describing leptin induced rapid

reorganization of synapses in the ARC (24).

Local regulation of mRNA translation plays an

important role in axon guidance and neuronal

plasticity, which are processes that involve

mTOR activity (25). Conceivably, the inhibition

of mTOR signaling may block leptin_s anorec-

tic effect by suppressing the hormone_s synap-
tic remodeling activity.

Other fuel-sensitive kinases have been im-

plicated in the hypothalamic control of energy

balance. Like mTOR, AMP-activated protein ki-

nase (AMPK) is regulated by intracellular AMP/

ATP ratios. However, in contrast to mTOR,

AMPK activity is increased during fuel defi-

ciency (26) and inhibited by leptin and nutrient

signals (27). AMPK overexpression in the hy-

pothalamus increases food intake and body

weight, and its down-regulation inhibits feed-

ing (27). Moreover, the activation of AMPK-

dependent mechanisms leads to the inhibition

of mTOR activity (28). Thus, AMPK and mTOR

may have overlapping and reciprocal func-

tions (Fig. 4D).

These fuel-sensitive signaling pathways

may ultimately provide important insights into

the link between obesity and type 2 diabetes. In

peripheral organs such as the liver, and in skel-

etal muscle, fuel overabundance is deleterious

because it alters the activity of fuel-sensitive ki-

nases (increased mTOR and decreased AMPK),

causing insulin resistance, which in turn sup-

presses nutrient uptake into tissues (10, 26). In

the CNS, an overabundance of fuel and

nutrients may produce similar changes in

mTOR and AMPK signaling, leading, con-

versely, to a beneficial reduction in nutrient

intake and in the level of stored fat. As long as

the CNS responses are adequate, the organism

can remain in a state of metabolic balance.

However, an imbalance between peripheral and

CNS fuel-sensing pathways may predispose

toward the development of obesity and/or

diabetes.

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D. Russell, J. Sorrell, and M. Toure for expert technical
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with artwork preparation; and H. Shi for assistance with
the quantification of the immunohistochemistry.
Supported by NIH grants DK 17844, DK 54080, and
DK 54890. G.T. receives research support from
Novartis.

Supporting Online Material
www.sciencemag.org/cgi/content/full/312/5775/927/DC1
Materials and Methods
Figs. S1 to S5
Table S1
References

21 December 2005; accepted 25 March 2006
10.1126/science.1124147

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12 MAY 2006 VOL 312 SCIENCE www.sciencemag.org930