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of Pkc- and insulin receptor signaling and
overt insulin resistance and glucose intolerance in the AccDKI mice, indicating that Ampk
phosphorylation of Acc has a crucial role in
reducing this lipid-induced cell-autonomous
insulin resistance (Fig. 1). The authors then
used hyperinsulinemic-euglycemic clamps to
show that the AccDKI mice had defects in the
suppression of hepatic glucose production by
insulin. Importantly, they demonstrated that
fat-induced insulin resistance and the requirement of Ampk and Acc in metformin action can
be modeled cell-autonomously in hepatocytes,
providing a useful system for future studies.
The study by Fullerton et al.4 carefully
delineates the role that AMPK and its originally defined substrates ACC1 and ACC2
have in reducing lipid stores in liver and
muscle, which, in turn, results in heightened
insulin sensitivity and reduced blood glucose levels. Given the recent evidence that
metformin can acutely lower hepatocyte
glucose production via direct effects of AMP
on adenylate cyclase13, the overall impact of
AMPK-independent versus AMPKdependent effects of metformin on distinct

metabolic pathways needs to be investigated

further across different dietary and metabolic conditions. Finally, although these
authors show that ACC phosphorylation is
required for metformin action4, this does
not preclude the possibility that other pathways independent of AMPK are required for
metformin to lower glucose, nor does it rule
out that mutation of other AMPK substrates
can also disrupt the ability of metformin to
restore aspects of glucose and lipid homeostasis in insulin resistance. For example, AMPK
phosphorylates multiple components of the
mammalian target of rapamycin complex 1
(mTORC1) pathway, which have key roles in
lipid metabolism and govern insulin sensitivity via effects on insulin receptor substrate
(IRS) and GRB10 protein families16. Whether
mTORC1 substrates, IRS1 or other AMPK
substrates are involved in the potent clinical
activity of metformin awaits future studies.
Given the vast number of patients taking
metformin and the burgeoning interest in
metformin use for polycystic ovary syndrome
and in cancer prevention trials, there is still a
lot to learn about the mechanisms driving the

effectiveness of this age-old drug in different

disease contexts.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
1. Witters, L.A. J. Clin. Invest. 108, 11051107
(2001).
2. Hundal, R.S. et al. Diabetes 49, 20632069 (2000).
3. Jornayvaz, F.R. & Shulman, G.I. Cell Metab. 15,
574584 (2012).
4. Fullerton, M.D. et al. Nat. Med. 19, 16491654
(2013).
5. Owen, M.R., Doran, E. & Halestrap, A.P. Biochem. J.
348, 607614 (2000).
6. El-Mir, M.Y. et al. J. Biol. Chem. 275, 223228
(2000).
7. Zhou, G. et al. J. Clin. Invest. 108, 11671174
(2001).
8. Foretz, M. et al. Diabetes 54, 13311339 (2005).
9. Yang, J., Maika, S., Craddock, L., King, J.A. & Liu, Z.M.
Biochem. Biophys. Res. Commun. 370, 248253
(2008).
10. Cool, B. et al. Cell Metab. 3, 403416 (2006).
11. Viollet, B. et al. Acta Physiol. (Oxf.) 196, 8198
(2009).
12. Foretz, M. et al. J. Clin. Invest. 120, 23552369
(2010).
13. Miller, R.A. et al. Nature 494, 256260 (2013).
14. Hardie, D.G., Ross, F.A. & Hawley, S.A. Nat. Rev. Mol.
Cell Biol. 13, 251262 (2012).
15. Steinberg, G.R. & Kemp, B.E. Physiol. Rev. 89,
10251078 (2009).
16. Mihaylova, M.M. & Shaw, R.J. Nat. Cell Biol. 13,
10161023 (2011).

Reassessing the role of astrocytes in ammonia

neurotoxicity
Tore Eid & Tih-Shih W Lee
High blood ammonia, as seen in severe liver disease and urea cycle disorders, is neurotoxic and difficult to treat.
A new study shows that the toxic effects are caused by impaired astrocyte potassium bufferingnot astrocyte
swelling, as previously thoughtand can be partially blocked by the diuretic bumetanide (pages 16431648).
Ammonia, a structurally simple yet biologically potent molecule, is extensively present
in nature and crucially involved in a variety of
processes ranging from industrial production
of fertilizers to pH regulation and metabolism
of nitrogen-containing compounds in living
organisms. In the human body, as much as
4 g of ammonia is formed every day, mainly
from the physiological action of bacterial
enzymes on colonic content and from hydrolysis of glutamine in the cells of the small and

Tore Eid is at the Departments of Laboratory

Medicine and Neurosurgery, Yale University School
of Medicine, New Haven, Connecticut, USA.
Tih-Shih W. Lee is at the Department of Psychiatry,
Yale University School of Medicine, New Haven,
Connecticut, USA.
e-mail: tore.eid@yale.edu

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large intestine by the enzyme glutaminase.

A smaller amount of ammonia is also synthesized in other organs via one of several biochemical routes involving enzymes such as
glutaminase, glutamic acid dehydrogenase and
adenosine deaminase.
In aqueous solutions, ammonia (NH3) is
in equilibrium with ammonium ion (NH4+).
Even though only 12% is found as NH3 at
physiological pH, the neutral form of the
molecule readily permeates most cell membranes, including those of the blood-brain
barrier. Thus, increased blood ammonia or
hyperammonemia rapidly leads to elevated
levels of the molecule in most organs, including the central nervous system (CNS). Because
hyperammonemia is toxic to the CNS1, several
metabolic pathways for clearing excess ammonia are in place. Quantitatively, the most important of these is the urea cycle or ornithine cycle,

which is highly active in liver cells2; intestinal

ammonia is absorbed via the portal circulation,
transported to the liver and metabolized into
urea, which is ultimately excreted in urine.
Other, less effective pathways exist, such as
the glutamine synthetase reaction in astrocytes
(Fig. 1)3. This reaction converts up to 25% of
blood-derived ammonia to glutamine in the
cytosol of astrocytes.
Humans with severe liver disease or children
with genetic defects in urea cycle enzymes often
exhibit hyperammonemia and signs of neurological impairment, such as poor appetite,
vomiting, reduced consciousness and seizures.
The glutamine synthetase pathway in these
patients is usually intact, but it is not sufficient
to prevent CNS toxicity. In fact, some studies suggest that the excess formation of brain
glutamine during hyperammonemia via this
pathway leads to a cascade of deleterious events

volume 19 | number 12 | DECEMBER 2013 nature medicine

news and views

Astrocyte

Axon terminal

2
GLN

5
GABA

Ca++
Volume

GS

6
H+ + NH3

NH4+

3
1A

NH3 + H+

NH4+

K+

1B
+

NH4

NH3 + H

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2013 Nature America, Inc. All rights reserved.

Microvessel

NH3 + H

Cl
GABAA

7
Cl

NKCC1
Cl
+
Bumetanide
+

10

+
+
+

Depolarization
and increased
excitability

NH4

Postsynaptic element

Figure 1 Uptake, metabolism and proposed toxic mechanism of ammonia in the CNS. At physiological
pH of 7.4, about 12% of blood ammonia is present as NH3, which readily enters the brain, either into
the perivascular astrocyte process (1A) or into the extracellular space (1B). In the astrocyte, ammonia
may be metabolized to glutamine (GLN) (2) via glutamine synthetase (GS). Rangroo Thrane et al.5
showed that during conditions of hyperammonemia, increased extracellular brain ammonium (NH 4+)
competes with astrocyte uptake of extracellular potassium (3), causing an increase in extracellular
potassium (4), astrocyte calcium (5) and astrocyte shrinkage (6). The increased extracellular potassium
and ammonium facilitate the flux of chloride into postsynaptic elements via NKCC1, leading to
increased intracellular chloride (7). Binding of GABA to GABAA receptors on the surface of the
postsynaptic element (8) causes flux of chloride out of the cell (9), membrane depolarization and
increased excitability (10).

that include astrocyte swelling, glutamate

toxicity and formation of reactive oxygen species4. In this issue of Nature Medicine, Rangroo
Thrane et al.5 show that increased ammonia
in mice does not lead to astrocyte swelling
but rather leads to impaired astrocyte buffering of potassium, altered -aminobutyric acid
(GABA)-mediated neurotransmission and
neurological dysfunction. They demonstrate
that the adverse neurological effects of ammonia can be partially attenuated by the US Food
and Drug Administration (FDA)-approved
diuretic bumetanide, offering new hope in the
treatment of ammonia neurotoxicity.
The authors assessed the behavioral,
molecular and electrophysiological effects of
hyperammonemia on normal mice and mice
lacking ornithine transcarbamylase, one of
the enzymes in the urea cycle5. Elevated blood
or brain ammonia found in either of these
animals resulted in neurological manifestations such as impaired learning, increased
motor activity and seizures. Rangroo Thrane
et al.5 then used awake mice to show that
increases in brain ammonia compromise the
ability of astrocytes to clear potassium from
the extracellular space, most likely via saturation of potassium uptake mechanisms by
increased ammonium ion levels (Fig. 1). This
is a notable finding because elevated extracellular potassium concentrations are known to

cause seizures in brain slice preparations 6.

Prior studies have also shown that patients
with one of the most common chronic seizure
disorderstemporal lobe epilepsyexhibit
impaired uptake of extracellular potassium by
astrocytes, suggesting that perturbed potassium homeostasis is also implicated in human
seizures7,8. The work by Rangroo Thrane et
al.5 provides the first evidence that directly
links excess ammonia to increased extracellular potassium and neurological dysfunction,
including seizures, in awake animals.
Earlier ex vivo studies have led to the conclusion that astrocyte swelling is a key component of ammonia neurotoxicity9. This notion
is further supported by the fact that nearly 80%
of individuals with hepatic encephalopathy,
a condition associated with hyperammonemia, develop brain edema10. Surprisingly,
in vivo two-photon laser scanning microscopy
showed that increased ammonia does not lead
to astrocyte swelling but rather leads to transient astrocyte shrinkage and increased astrocyte calcium signaling5 (Fig. 1). Astrocyte
swelling or brain edema only occurred in the
terminal stages of ammonia toxicityafter the
neurological changes had been established
arguing against astrocyte swelling as an early
(causal) event in ammonia toxicity. A possible explanation for this paradoxical finding
is that most experimental studies of ammonia

nature medicine volume 19 | number 12 | DECEMBER 2013

toxicity have used ex vivo preparations, which

may not represent the true biological milieu
of an intact, awake brain. However, although
brain edema occurs frequently in late stages
of hepatic encephalopathy in humans, it is
also seen earlier in the disease11. Moreover,
isolated cases of acute hyperammonemia
in humans can lead to rapid (within hours)
swelling of the brain12. The discrepancy in
swelling between the human cases and the
study by Rangroo Thrane et al.5 is not clear
but may be due to more complex pathological
alterations, possibly involving factors other
than ammonia in humans.
The authors next investigated the consequences of increased ammonia and compromised astrocyte potassium buffering on
GABA-mediated neurotransmission5. In the
adult brain, binding of GABA or drugs such as
benzodiazepines and barbiturates to GABAA
receptors on the surface of neurons causes a
flux of chloride ions (Cl) into the cells, resulting in hyperpolarization of the cell membrane
and decreased neuronal activity (inhibition).
Chloride flows into the cells because the
intracellular concentration of the ion is lower
than the extracellular concentration. A chloride shift with increased amounts of chloride
inside neurons is seen during brain development and in pathological conditions such as
epilepsy13. Stimulation of GABAA receptors in
this context will lead to a flux of chloride out
of the cell with depolarization and possibly
increased neuronal activity (excitation). The
chloride shift is largely driven by Na+ K+ 2Cl
transporter type 1 (NKCC1), which pumps
two Cl into the cell along with one molecule
each of Na+ and K+.
Rangroo Thrane et al.5 found that the
elevations in extracellular ammonium and
potassium ions resulted in overactivation of
NKCC1 on cortical pyramidal cells, causing
elevated intracellular Cl (Fig. 1) and depolarization upon binding of GABA to the cell.
Interestingly, blockade of NKCC1 with the
FDA-approved diuretic bumetanide led to
increased survival and reduction in neurotoxic behaviors, including myoclonic seizures,
after exposure to ammonia. Prior studies have
shown that bumetanide stops seizures in neonatal models of epilepsy and in case reports of
human pediatric seizures14. Thus, inhibition of
NKCC1 may be a new and promising approach
for treating ammonia neurotoxicity and certain
types of seizures. However, the use of NKCC1
inhibitors for these conditions requires careful
testing, possibly involving more CNS-specific
drugs. This precaution is partly due to the fact
that bumetanide, which has potent systemic
effects, may precipitate hepatic encephalopathy in patients with liver disease.
1573

news and views

Competing financial interests
The authors declare no competing financial interests.

10. Blei, A.T. Hepatology 13, 376379 (1991).

11. Kumar, R. et al. J. Magn. Reson. Imaging 27,
10611068 (2008).
12. Pillai, U., Kahlon, R., Sondheimer, J., Cadnapaphorncai, P.
& Bhat, Z. JPEN J. Parenter. Enteral Nutr. 37,
134137 (2013).
13. Ben-Ari, Y., Khalilov, I., Kahle, K.T. & Cherubini, E.
Neuroscientist 18, 467486 (2012).
14. Dzhala, V.I. et al. Nat. Med. 11, 12051213
(2005).

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2013 Nature America, Inc. All rights reserved.

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2. Krebs, H.A. & Henseleit, K. Z. Phys. Chem. (N.F.) 210,
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3. Martinez-Hernandez, A., Bell, K.P. & Norenberg, M.D.
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4. Albrecht, J. & Norenberg, M.D. Hepatology 44,
788794 (2006).

5. Thrane, R. et al. Nat. Med. 19, 16431648

(2013).
6. Traynelis, S.F. & Dingledine, R. J. Neurophysiol. 59,
259276 (1988).
7. Bordey, A. & Sontheimer, H. Epilepsy Res. 32,
286303 (1998).
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814825 (2012).
9. Norenberg, M.D., Jayakumar, A.R., Rama Rao, K.V. &
Panickar, K.S. Metab. Brain Dis. 22, 219234 (2007).

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