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Discovery at the VICB

 

 

 

 

 

 

Controlling Energy Balance

 

By: Carol A. Rouzer, VICB Communications
Published:  February 9, 2015

 

 

A newly discovered link between the melanocortin-4 receptor and a potassium channel sheds light on the regulation of food intake and energy expenditure.

 

As its toll becomes increasingly apparent, the obesity epidemic is rapidly emerging as the world’s number one public health crisis. A potentially important therapeutic target in the battle against obesity is the melanocortin-4 receptor (MC4R). Expressed in over 100 regions in the brain, the MC4R is found in particularly high concentrations in the paraventricular nucleus (PVN) of the hypothalamus, where it plays a major role in the regulation of energy homeostasis and appetite. Key ligands for the receptor include α-melanocyte stimulating hormone (α-MSH), and agouti-related protein (AgRP), both of which are produced in the arcuate nucleus of the hypothalamus by neurons which project to MC4R-expressing neurons in the PVN. α-MSH acts as an agonist at MC4R, triggering signaling via G proteins linked to Gαs. α-MSH-mediated MC4R activation leads to increased energy expenditure and a reduction in food intake (Figure 1). AgRP competes with α-MSH for MC4R binding and blocks tonic activity of the receptor. Its antagonistic activity results in decreased energy expenditure and increased food intake. The role of MC4R in regulating energy balance suggests that agonists of the receptor should serve as anti-obesity therapeutic agents. However, attempts to exploit this target have been thwarted by unwanted side effects, particularly hypertension. Now, Vanderbilt Institute of Chemical Biology member Roger Cone, research instructor Masoud Ghamari-Langroudi, and their collaborators Glenn Millhauser (University of California, Santa Cruz), Helen Cox (King’s College London), and Jerrod Denton (VICB) report a new mechanism for MC4R-dependent signaling that may pave the way for drugs that suppress appetite with fewer side effects [M. Ghamari-Langroudi, et al. (2015) Nature, published online January 19, doi:10.1038/nature 14051].

 

 




Figure 1.
Figure 1. Role of MC4R in energy homeostasis. Neurons in the arcuate nucleus of the hypothalamus produce pro-opiomelanocortin (POMC, the precursor to α-MSH) or AgRP. Projections of these neurons to the PVN reach MC4R-expressing neurons where α-MSH activates MC4R, leading to increased energy expenditure and decreased food intake. Antagonism by AgRP has the opposite effects.

 

The investigators began with a study of exactly how α-MSH and AgRP affect the firing of MC4R-expressing neurons in the PVN. They found that α-MSH increased the frequency of firing by depolarizing the neurons, while AgRP had the opposite effect. Careful pharmacologic studies showed that these effects on neuron firing and membrane potential were not dependent on G protein-dependent signaling. Electrophysiological studies of the relationship between current and voltage in the MC4R-expressing PVN neurons revealed that α-MSH decreased membrane conductance and generated a current that was inwardly rectifying, meaning that potassium ions could flow through the channel into the cells faster than out of the cells, depending on the membrane potential. AgRP exerted the opposite effect on membrane conductance and current flow. These observations suggested that both α-MSH and AgRP were acting on an inwardly rectifying potassium channel (Kir), but with opposite effects.

 

In resting cells, Kir channels allow a slow efflux of potassium ions out of the cell to help maintain the membrane potential. The investigator’s findings suggested that α-MSH was blocking this outflow, resulting in a reduction in membrane conductance and a net current of positive ions into the cell (Figure 2). In contrast, AgRP appeared to increase flow through this same channel. Reducing intracellular Mg2+ or increasing phosphatidyl inositol 4,5-bisphosphate magnified the α-MSH-induced current, observations that were consistent with the involvement of a Kir channel. Pharmacologic studies using selective inhibitors provided by the Denton laboratory identified Kir7.1 as the channel most likely blocked by α-MSH. This conclusion was supported by the fact that 90% of PVN neurons that express MC4R also express Kir7.1.

 

 

 

 


Figure 2.
Effect of MC4R signaling on Kir7.1 channels. POMC neurons in the arcuate nucleus produce α-MSH, which activates MC4R in PVN neurons. The conventional model of MC4R signaling (center orange box) posits that α-MSH binding to MC4R results in a Gαs-mediated increase of cAMP. This pathway is antagonized by AgRP, which competes for MC4R binding with α-MSH. The present work suggests that binding of α-MSH to MC4R leads directly to blockade of Kir7.1, which decreases the flow of potassium out of the cell, resulting in membrane depolarization and increased excitability (right). This effect does not require a G protein. AgRP can also bind directly to MC4R, which leads to increased flow of potassium ions through Kir7.1, resulting in membrane hyperpolarization and decreased excitability (left). This effect also does not require a G protein, and is not the result of blocking the binding of α-MSH. Image reproduced by permission from Macmillan Publishers Ltd: Nature, M. Ghamari-Langroudi, et al. (2015) published online Jan. 19, DOI: 10.1038/nature14051. Copyright 2015.

 

 

To further explore the effect of MC4R-dependent signaling on Kir7.1 function, the investigators transfected genes for both the receptor and the potassium channel into HEK293 cells. They then used the transfected cells to develop a direct assay of MC4R-dependent modulation of Kir7.1 conductance by employing a Tl+-sensitive fluorescent dye that passes readily through the channel. The assay revealed that α-MSH decreased channel conductance with an IC50 of ~32 nM, while AgRP increased conductance with an EC50 of ~2.5 nM. Of particular interest was their finding that the effects of AgRP on the channel did not appear to be mediated through inhibition of α-MSH binding but rather via a direct effect of AgRP on the receptor. They also further confirmed that neither ligand required a G protein to exert its effects on Kir7.1 conductance.

 

The AgRP analog miniAgRP has the same affinity as AgRP for MC4R and the same ability to block G protein-dependent signaling by α-MSH. The investigators found, however, that miniAgRP lacks the ability to stimulate Kir7.1-mediated conductance or to potentiate food intake in rats. Similarly, the α-MSH analog MC4-NN2-0453 was unable to stimulate G protein-dependent signaling by MC4R, but efficiently coupled the receptor to Kir7.1. This analog potently suppressed food intake in mice. These findings suggest that regulation of appetite by MC4R occurs through coupling of the receptor to Kir7.1 rather than through G protein-dependent signaling. The results also suggest that the two signaling pathways can be pharmacologically separated. The observation that MC4-NN2-0453 reduced food intake without increasing blood pressure in the mice suggests that selective modulation of MC4R-depending signaling through Kir7.1 is a promising new approach to suppressing appetite in vivo.

 

 

 

 

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