atp sensitive potassium channel mediated lactate effect
TRANSCRIPT
Sachin Mehta
Analysis of article: “ATP-Sensitive Potassium Channel-Mediated Lactate Effect on Orexin Neurons: Implications for Brain Energetics during Arousal” Matthew P. Parsons and Michiru Hirasawa. 2010. The Journal of Neuroscience. 30(24): 8061-8070.http://www.jneurosci.org/content/30/24/8061.full
Introduction
The brain utilizes a significantly large proportion of glucose in
the body. While it is still not entirely confirmed, the widespread belief
is that glucose uptake is performed by certain “glucosensing neurons”
in the hypothalamus and brainstem (Levin et al., 2004). They
maintain this homeostasis by excitatory or inhibitory effects.
However, these cells are not the only glucose-metabolizing cells in the
brain. Astrocytes have been identified as the primary cell type to
utilize glucose, and they produce lactate as an additional substrate.
In the last decade or so, new evidence has emerged suggesting
that lactate is one of the primary sources of energy and regulates
certain metabolic factors. The extent of its role in certain areas of the
brain is not completely understood. Parsons and Hirasawa conducted
their research on a group of cells known as orexin neurons, which are
known to play an important part in food intake, autonomic function
and wakefulness. These qualities make them great candidates to
consider, as they obviously require energy input and metabolism for
function. Previous research has shown that orexins are stimulated by
glucose and lactate, and perhaps require this lactate as their
fundamental source of energy. This study showed that orexin neurons
sense certain levels of astrocyte-produced lactate, and in turn,
modulate their neuronal activity and responses to these levels.
Experimental System
This research used a combination of electrophysiology on rat
and mice brains, immunohistochemistry, data and drug analyses to
learn more about orexin neurons. The rats and mice were
anesthetized and decapitated to obtain coronal hypothalamic slices.
Glucose concentrations were measured using patch-clamp recordings
on the hypothalamic slices. Additional patch-clamp tests were used
to measure the firing (action potential) characteristics of the orexin
neurons. These values were used to deduce orexin neuron
concentration in comparison to the high presence of melanin-
concentrating hormone (MCH) in the same area. Phenotypic
characterizations of the orexin neurons were performed by voltage
ramps and current injections.
Immunohistochemical techniques were performed to detect
antibody signaling at different parts of the sectioned brains. Rabbit
anti-MCH and goat anti-orexin A antibodies were mixed together to
determine the localizations of certain orexins and MCH. Additionally,
KATP (ATP-sensitive potassium) channel subunits were identified
using rabbit anti-Kir6 antibodies. Immunofluorescence was then
visualized using confocal microscopy.
Data analysis was an important part of this study. Researchers
used Synaptosoft software to analyze certain action potential
properties, such as current, frequency and membrane potential. T-
tests were also performed in order to deem the significance of the
data.
Finally, a variety of drugs were used in conjunction with the
electrophysiology techniques in order to understand structures and
functions of the key players in orexin neuron metabolism.
Experiments and Results
In order to determine the role of lactate and whether or not it
was preferred by orexins, cell-firing experiments showed the necessity
for glucose and lactose. 4-CIN, an inhibitor of MCT’s
(monocarboxylate transporters--needed for lactate transport across
membrane) was used, and showed an inhibition of firing activity.
Next, the behavior of orexins in a glucose-free environment was
shown to completely shut off firing activity, revealing the true
importance of glucose as an energy substrate. Lactate supply was
then used to bring back complete firing activity. The mechanism by
which this occurs is unclear, as it is possible that the lactate produced
by nearby astrocytes was enough for this restoration. The
researchers then tested the necessity of endogenous astrocyte-
produced lactate on the orexin neurons. Hypothalamic slices were
drained of all remaining glucose and other energy substrates by glial
toxin fluoroacetate (FAC), and the firing rate was examined in
conjunction with a lactate and glucose supply. The results showed
that astrocytes convert the glucose to lactate. This lactate is then
used by orexins and is responsible for maintaining any spontaneous
firing activity.
KATP channels were examined because they are known to be
crucial in lactate transport. Their structures were investigated using
immunofluorescence labeling. These techniques show that KATP
channels of the orexin neurons consist of Kir6.1 and SUR1 subunits,
which are modulated depending on the specific metabolic activities of
the cells. Glibenclamide blocked the hyperpolarization of the KATP
channels, indicating that these channels are necessary for firing
activity and that KATP channels control lactate levels.
Lastly, the researchers determined that these orexin neurons
are capable of acting as “lactate sensors.” They tested this idea by
depleting all lactate from the hypothalamic slices, and testing the
firing rate. The orexins based their firing rates on the level of lactate
available, implying that their activity is concentration-dependent.
Conclusion
The research performed by Parsons and Hirasawa showed that
lactate produced by astrocytes is needed and preferred as an energy
source by orexin neurons. Because lactate seemed to induce an
excitatory effect on the firing frequency, it was concluded that orexin
neurons act as concentration-dependent lactate sensors. That is,
these neurons can perceive a change in the concentration of
extracellular lactate levels, which can alter their cellular effects and
expression. Lactate also plays a crucial role in sustaining a normal
resting membrane potential when combined with glucose and KATP
current. Lastly, the researchers concluded that orexin neurons
contain a relatively generous amount of intracellular lactate, which
serves as an endogenous energy supply in the absence of glucose.
Significance
This study sheds light on the importance of lactate as an energy
substrate and a paracrine factor. It is capable of providing signaling
effects to orexin neurons, indicating brain activity and energy supply.
High lactate levels result in stimulated orexin and KATP channel
activity. Furthermore, orexin neurons are essential in the astrocyte-
coupling process and can provide additional neuroprotection.
Because orexin neurons play a significant role in the physiology of
certain processes, such as wakefulness and food intake,
understanding even more thoroughly how and what purpose lactate
serves the brain will provide us with a key to treating GI disorders
and sleep pattern phases (Shram et al., 2002). Finally, since this area
of study is relatively new, much more research is needed to truly
understand the extent and importance of lactate processes in the
brain.
Literature Cited
Levin BE, Routh VH, Kang L, Sanders NM, Dunn-Meynell AA
(2004).
Neuronal glucosensing: what do we know after 50 years?
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53:2521–2528.
Shram N, Netchiporouk L, Cespuglio R (2002). Lactate in the brain of
the
freely moving rat: voltammetric monitoring of the changes
related to the
sleep–wake states. Eur J Neurosci 16:461– 466.