Date: March 4th, 2010

Speaker's Name and Affiliation:

• Matthew Hayes
• Psychology and Neuroscience University of Pennsylvania Philadelphia

Seminar Title: "Hindbrain control of GLP-1 receptor-mediated effects on energy balance"

Chair: Harry R. Kissileff, Ph.D.

Rapporteur: Kathleen L. Keller, Ph.D.

Attendees and their Affiliation:

Kathleen Keller	Columbia/Obesity Research Center
Harry Kissileff	Obesity Research Center
JA Grinker	University of Michigan
Andras Hajnal	PSU - C Hershey
Annemarie Olsen	NYORC
Karen Ackroff	Brooklyn College
Michael Lewis	Hunter/Columbia
B Balkan
Gerry Smith	WMCM
Changwon Kim	St. Luke's
Gary Schwartz	Einstein
John Glendinning	Barnard
Joe Vasselli	Obesity Research Center
Jennifer Gillman	Barnard
Susan Carnell	NYORC
Charlesa Gibson	NYORC
Allan Geliebter	NYORC

Summary: (Prepared by the Rapporteur)

The peripheral and central glucagon-like-peptide-1 (GLP-1) systems play an essential role in glycemic and energy balance regulation. Thus, pharmacological targeting of peripheral and/or central GLP-1 receptors (GLP-1R) may represent a potential long-term treatment option for both obesity and type-II diabetes mellitus (T2DM). Uncovering and understanding the neural pathways, physiological mechanisms, specific GLP-1R populations, and intracellular signaling cascades that mediate the food intake inhibitory and incretin effects produced by GLP-1R activation are vital to the development of these potential successful therapeutics. Particular focus is devoted to the essential role of the nucleus tractus solitarius (NTS) in the caudal brainstem, as well as the gut-to-brain communication by vagal afferent fibers in mediating the physiological and behavioral responses following GLP-1R activation.

GLP-1, a posttranslational product of proglucagon, is a neuropeptide that is endogenously released principally from two distinct sources: 1) "L" cells in the gastrointestinal (GI) tract following nutrient entry; and 2) neurons of the nucleus tractus solitarius (NTS) in the caudal brainstem that project to GLP-1 receptors (GLP-1R) both locally and throughout the brain. Exogenous stimulation of either peripheral or central GLP-1Rs engages a set of physiological responses that include reduced food intake, inhibition of gastric emptying, and increased glucose-stimulated insulin secretion. One neuronal population within the CNS clearly stands out in mediating the intake inhibitory effects of both the peripheral and central GLP-1 systems: the NTS. This talk discusses the mechanisms mediating the intake inhibitory effects that follow activation of both peripheral and hindbrain GLP-1Rs, with special emphasis focused on the role of the NTS and the gut-to-brain communication by vagal afferent fibers in GLP-1R-mediated physiological and behavioral responses.

Data discussed examine the endogenous contribution of NTS GLP-1R activation to the normal physiology of food intake control given that: 1) NTS neurons are the endogenous source of CNS GLP-1; 2) caudal brainstem processing is sufficient to mediate suppression of intake by hindbrain GLP-1R activation; 3) the NTS receives and integrates both vagal afferent satiation and blood born energy status signals and issues output commands essential to energy balance control; and 4) gastric distention activates GLP-1 containing neurons in the NTS. Findings reported show that the intake suppressive effects that follow ingestion of a preload (9ml of a nutritionally complete liquid meal, Ensure) require endogenous hindbrain GLP-1R activity, as both 4th icv and direct NTS delivery of the GLP-1R antagonist, Exendin-(9-39), increased food intake following ingestion of this preload. Ingestion of the Ensure preload gives rise to an array of satiation signals from the GI tract, including stomach distension and intraduodenal nutrient contact, each of which excite vagal afferents projecting to NTS neurons. Thus, subsequent tests determined whether endogenous hindbrain GLP-1R activity mediates suppression of intake from gastric distension or intraintestinal nutrient infusion. Blockade of hindbrain GLP-1R attenuated the suppression of intake by gastric distension but did not affect the intake suppressive effect of intraduodenal nutrient infusion. Taken together, these findings indicate that endogenous NTS GLP-1R activity contributes to the endogenous control of food intake by mediating the satiating effects of gastric distension.

Having established that the NTS GLP-1R are involved in the normal control of food intake, the intracellular signaling cascades mediating this intake suppressive effect were subsequently examined both behaviorally in vivo and in vitro from tissue lysates of the dorsal vagal complex. The collective set of findings show that gastric distensiongenerated vagal afferent signaling activates hindbrain GLP-1R leading to a suppression in food intake. This suppression of food intake occurs through a reduction in meal number, not through a change in meal size. This finding is in opposition to the suppression of intake by peripheral GLP-1R ligand administration, where the intake suppression occurs through a reduction in meal size, not meal number. The intracellular signaling pathways mediating the intake suppression by NTS GLP-1R activation occurs through a coordinated PKA-mediated suppression of AMPK activity and PKA-dependent activation of p44/42 MAPK/MEK signaling. These signaling responses within GLP-1R expressing neurons likely produce long-term (>24h) intake and body weight suppressive responses through CREB-mediated transcriptional effects, thus allowing NTS neurons to integrate various anorectic signals involved in meal-to-meal food intake control.

Question & Answers:

Q. In terms of the signals for energy availability, are there receptors in the NTS for them?
A. For leptin, yes. But for some of the other signals, it is debatable.

Q. Margolskee's group has found evidence that GLP-1 is released in the duodenum. Do you think the amount is released is too low to be clinically relevant?
A. This group has also published that chemoreceptors in the oral cavity also released GLP-1. They think it might be involved in the cephalic phase response. I'm not sure if they can determine in vivo if this is clinically relevant or not.

Q. Where does GLP-1 degradation occur?
A. It occurs in multiple levels. In enterocytes, liver, brain. Only about 1-2% of GLP- 1 that is secreted from the intestine actually makes it into general circulation.

Q. Is the GLP-1 agonist given orally?
A. Exendin-4 is given by IP injection.

Q. Is there data on the development of tolerance or resistance to these medications (GLP-1 agonists)?
A. For GLP-1, there is little development of tolerance over time.

Q. How do the levels of GLP-1 infusions in these studies compare to physiological levels?
A. Exendin-4 is given in pharmacological doses.

Q. Have people compared the effectiveness of Exendin-4 given via different routes of administration?
A. Yes, I am doing that right now and other labs have also tested this. Also, I'm sure that the pharmaceutical companies have these data, but may not have published them yet.

Q. Is GLP-1 peripheral, or does it penetrate the brain?
A. Yes, it penetrates the brain, but the part that penetrates the brain appears to not have any physiological action.

Q. Does peripheral GLP-1 administration increase central GLP-1 release?
A. I'm not sure anyone has looked at that. It's a good question.

Q. Do you see a difference in potency between giving peripheral GLP-1 or Exendin- 4?
A. Yes. Exendin-4 is far more effective.

Q. Is there a rebound effect?
A. We do see a rebound if we wait long enough. I've begun to avoid using high administration doses to avoid this.

Q. Do you use powdered food in your feedometer?
A. No, in the current feedometers reported with these studies we are using pellets, but this eventually became a problem because the feedometer had to be continually updated and recalibrated to adjust for the change in vector caused by the rat pulling on the food hopper attached to the load cell.

Q. Do you think that peripheral GLP-1's effect is mediated by changes in gastric emptying?
A. I believe that gastric emptying plays a role, but does not explain the entire effect.

Q. Are you saying that there is no way to get meal size reductions after central GLP-1 injections?
A. No, I wouldn't say that, but under different GLP-1 doses, you might see different effects in meal size.

Q. Do you know why the effect there was delayed?
A. I don't know. I can say that if we test fasted animals, we do see an effect within the first half hour.

Q. When are you giving the drug?
A. Just before the dark cycle.

Q. Are the animals actively losing weight?
A. Yes.

Q. Why did you give a liquid preload first?
A. I've done it without a preload (in both deprived and non-deprived states) and I haven't seen an effect unless we given this small liquid Ensure drink first.

Q. What made you think of priming the animals with Ensure?
A. This came after a study by Diane Williams where she found a very similar effect in the periphery.

Q. Have you measured GLP-1 response to the preload?
A. We haven't done that yet.

Q. Have you tried sham feeding Ensure?
A. We haven't tried that yet.

Q. Did you check to see if nearby neurons were activated by peripheral GLP-1 injection?
A. That is a good suggestion. I wasn't able to see any response to ligand administration in adjacent nuclei to the medial subnucleus of the NTS.

Q. So gastric distension alone is sufficient to release GLP-1?
A. We have not measured central GLP-1 release.

Q. What about trying a pyloric cuff with Ensure ingestion?
A. That is a good suggestion. We haven't tried that yet.

Q. You are getting effects in vitro at 30 minutes, but in vivo you did not get changes in food intake until 3 hours?
A. Right, there are differences in the time course of these events and it is a paradox.

Q. Don't most anorectic drugs decrease meal size and not meal number?
A. Yes, but it depends on which system you activate. Here we are targeting the central GLP-1 system; which as I showed produces a different response (suppression in meal number) from that seen with peripheral GLP-1R ligand administration (suppression in meal size).

Q. Have you looked at PYY? Are GLP-1 and PYY co-expressed?
A. We have not looked at this. They are not co-expressed in the NTS, but they are co-secreted in the L-cells in the small intestine.