Project Summary The proposed research of this R01 application focuses on creating a second-generation glucagon-like peptide- 1 (GLP-1)-based pharmaceutical that retains all of the blood glucose lowering profiles but completely eliminates the major side effects of nausea, vomiting and malaise. Such side effects, along with hypophagia, are produced by existing GLP-1R agonists due to central nervous system (CNS) penetrance and direct action in the brain. Thus, we sought to create GLP-1R agonists with reduced brain penetrance but with the full potent pharmacodynamic profile on pancreatic GLP-1R populations. Our novel preliminary data convincingly demonstrate the ability of a vitamin B12 (B12) conjugate of the GLP1-R agonist Exendin-4 (Ex4), namely (B12- Ex4), to produce hypoglycemia in a glucose tolerance test (GTT) in both mouse and rat models without producing hypophagia or nausea/malaise. This glucoregulation without nausea/malaise appears to be due to a virtual absence of ligand penetrance into the CNS, a hypothesis supported by radiolabelled B12 studies showing extremely low levels of B12 entry into the brain, as well as immunohistochemical analyses of fluorescently- tagged B12-Ex4 in comparison to native Ex4 at the site of CNS activation. This application therefore tests the following specific aims to both enhance the preclinical assessment of B12-Ex4 as a second-generation T2DM therapeutic and test the hypothesis that the total portfolio of therapeutic effects that exploit the `B12-family' can be enhanced by conjugation of Ex4 to a fragment of B12, specifically cobinamide (Cbi), which targets Haptocorrin (HC), a Cbi binding protein found only in mammals, including humans: [1] Characterizes the physiological, behavioral and anatomical mechanisms mediating the hypoglycemic effects of B12-Ex4 without the incidence of hypophagia and nausea/malaise using both lean/euglycemic and obese/hyperglycemic rat and mouse models; [2] Conducts systematic in vitro and in vivo analyses of a novel Cbi-Ex4 compound for glucoregulation without eliciting nausea/emesis or competing with endogenous B12 transport in the musk shrew which both is capable of emesis and expresses HC, like the human, but unlike the mouse and rat. The compounds described herein also offer scope to investigate, through relatively simple experimentation, the role of CNS versus periphery in the function of GLP-1R agonists in animals and humans. As such they have significant potential as investigative research probes in addition to their clear potential as a new generation of therapeutics. Most notably, these highly innovative experiments will provide the necessary pre-clinical analyses for the B12- and/or Cbi-based conjugates of Ex4. Such studies will lead to significantly improved, clinically beneficial, second generation therapeutics for the treatment of T2DM without the most common nausea/malaise side effects of existing GLP- 1-based therapeutics, which will improve patient quality of life,...