Role of the hormone LEAP2 in eating, body weight, blood glucose and survival

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PROJECT SUMMARY The hormone acyl-ghrelin serves as a key regulator of eating, body weight, blood glucose, and survival upon binding to its receptor, GHSR. In the past project period, we studied regulation of ghrelin secretion. A major finding was that under a severe caloric restriction protocol modeling starvation, ghrelin cell-expressed 1- adrenergic receptors (1ARs) mediate ghrelin secretion, which in turn defends against marked hypoglycemia and mortality. Also, following the recent identification of liver-enriched antimicrobial peptide 2 (LEAP2) as an endogenous GHSR antagonist, we extended our studies to characterize LEAP2 function at the cellular level and to determine plasma LEAP2 changes due to various metabolic perturbations in human subjects and mice. We found that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons, suggesting that LEAP2 serves as both a GHSR inverse agonist and antagonist. Also, we found that plasma LEAP2 is regulated by metabolic status: its levels increase with obesity and rising blood glucose and decrease with fasting and weight loss. These changes were mostly opposite of those of acyl-ghrelin. Collectively, this led us to propose the following model of LEAP2 function: 1) In obese states, LEAP2 rises and acyl-ghrelin falls, shifting the plasma LEAP2/acyl-ghrelin molar ratio higher and thus limiting acyl-ghrelin’s capacity to worsen obesity and glucose intolerance by raising food intake, body weight and blood glucose. 2) In nutritionally deficient states, such as that induced by severe caloric restriction, a fall in LEAP2 creates an environment in which elevated acyl-ghrelin can most effectively act to prevent life-threatening hypoglycemia. In the current R01 proposal, we test this model in 3 aims by using a combination of mostly novel tools to delete, knockdown, and/or neutralize LEAP2 in settings of obesity and severe caloric restriction. In Aim 1, we test whether deleting or blocking LEAP2 will exacerbate obesity and glucose intolerance in obesogenic settings, and we determine the extent to which LEAP2 and ghrelin gain access to different CNS regions. In Aim 2, we test whether under a severe caloric restriction protocol modeling starvation, deleting LEAP2 will further enhance the capacity of activated GHSRs to boost blood glucose and survival. In Aim 3, we delete LEAP2 selectively from liver or intestine – the two predominant sources of LEAP2 – and assess changes in plasma LEAP2 and metabolism in response to long-term high fat diet exposure and severe caloric restriction. We will use a collection of four new, unpublished recombinant mouse lines that allow us to delete or site-selectively delete LEAP2 and/or ghrelin, together with a novel viral vector that induces knockdown of LEAP2 expression, and a LEAP2 neutralizing monoclonal antibody. Our studies will provide fundamental insight into the functional significance of the recently characterized GHSR antagonist and inverse agonist LEAP2 and the related GHSR agonist acyl- ghrelin in the development of obesity and glucose intolerance under settings of nutritional overabundance and in the development of life-threatening hypoglycemia under settings of severe caloric restriction.