br Acknowledgments We would like to acknowledge

Acknowledgments We would like to acknowledge Jenna Hall, Marita Rivir, Kathleen Smith, Joyce Sorrell, and Emily Yates for assistance with in vivo pharmacology studies. We would like to acknowledge Joe Chabenne and Steph Mowery for assistance with in vitro cell-based assays, and Patrick J. Knerr for assistance with chemical synthesis. This work was partially supported by funds of the University of Cincinnati-College of Medicine (F102150) to D.P.-T.
Introduction Treatment of type 2 diabetes mellitus (T2DM) with glucagon-like peptide-1 receptor agonists (GLP-1RAs) leads to better glycaemic control, reduced body weight, and improvement in several cardiovascular risk factors, which has been demonstrated to be accompanied by improved micro- and macrovascular outcomes [1]. These benefits are mediated by the GLP-1R, a member of the class B family of G protein-coupled receptors, that is expressed in pancreatic beta-cells, various cell types of the gastrointestinal tract, and neurons throughout both the central (CNS) and the peripheral nervous systems [2]. Activation of GLP-1R signaling by GLP-1RAs improves glucose control by enhancing glucose-stimulated insulin secretion [3], [4], delaying gastric transit [5], [6], and decreasing plasma glucagon levels [7], and reduces body weight by activating anorexigenic pathways in the bazedoxifene sale [8]. Due to the glucose-dependence of beta-cell activation, which involves a number of intracellular mediators, includiing calcium and Epac2 [9], [10], GLP-1RAs are not associated with increased risk of hypoglycaemia [11]. While the broad metabolic benefits of GLP-1RAs have established this class in the T2DM treatment paradigm, many patients do not reach their glycaemic targets, and weight loss achieved with these agents remains well below what can be attained with bariatric surgery, the most potent clinical intervention for obesity [12]. Thus, there are significant opportunities to improve upon the existing GLP-1RA class. One emerging approach is to combine foundational GLP-1RA therapy with pharmacological strategies targeting additional pathways implicated in nutrient and energy metabolism, such as glucose-dependent insulinotropic polypeptide (GIP) [13]. GIP is an incretin that is secreted from K cells in the upper small intestine in response to food [14]. Postprandial GIP levels are approximately 4-fold higher compared to GLP-1 under normal physiological conditions [15]. GIP is responsible for the majority of the insulinotropic incretin effect in man [14], [16] and has important additional functions that are distinct from GLP-1. Unlike GLP-1, GIP is both glucagonotropic and insulinotropic in a glycaemic-dependent manner, dose-dependently stimulating glucagon secretion under hypoglycaemic conditions and insulin under hyperglycaemic conditions [17], [18], [19], [20], [21]. Although both GIPR and GLP-1R are present in beta-cells, GIPR expression is distributed differently in extra-pancreatic tissues as GIPR is abundant in adipose tissue [22] and is found in many non-overlapping areas of the CNS [23]. The biological activity of GIP on adipocytes has been investigated for some time, and although rather disparate, GIP is implicated in adipose tissue carbohydrate and lipid metabolism by its actions to regulate glucose uptake [24], lipolysis [25], and lipoprotein lipase activity [26], [27], [28], some of which support a role of GIP in fat accumulation that align with studies indicating GIPR null mice are resistant to obesity. Further, acute GIP infusion to humans increases adipose tissue blood flow [29], suggesting additional mechanisms of actions. Chronically elevating GIP levels in a transgenic mouse model has been shown to reduce diet-induced obesity (DIO) and improve insulin sensitivity, glucose tolerance, and beta-cell function [30]. These findings suggest that pharmacological activation of GIPR may have a therapeutic benefit on peripheral energy metabolism. In support of this, studies in DIO mice showed the body weight lowering effects of GLP-1RA are enhanced upon co-administration of a long-acting GIP analogue [31]. However, the role of GIP in the regulation of body weight has been controversial. Observations from genetically modified mice are confounding as both GIPR-deficient [27] and GIP-overexpressing mice [30] seem to be protected from obesity. In addition, chronic administration of a long-acting GIP molecule in rodents has no effects on body weight [31]. In the brain, GIP appears to activate neurons distinct from GLP-1, and central infusion of GIP in mice can inhibit food intake in a manner that is additive to GLP-1 [32]. Historically, the therapeutic utility of GIP has been limited by the fact that the incretin response to GIP is severely blunted in T2DM, possibly due to downregulation of the GIPR by high circulating glucose. A substantial body of data suggests, however, that GIP resistance can be largely overcome by agents that lower circulating glucose levels [33], [34], paving the way for considerations of GIP as add-on to glucose-lowering therapies, like GLP-1.