Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to GLP-1 receptor (GLP-1R) agonism in patients with type diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine if GIPR agonism contributes, we compared the effect of tirzepatide in obese wild-type and Glp-1r null mice.In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino (BCAAs) and keto-acids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent andindependent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Experimental models and experimental designsAll procedures were approved by the Institutional Care and Use Committee of the University of Pennsylvania and Eli Lilly and Company. Adult male C57BL/6 mice (Taconic) weighing ~20g at arrival (n=84) were housed under a 12-hour:12-hour light/dark cycle in a temperature-and humidity-controlled vivarium. Mice were individually housed in standard cages with ad libitum access to chow diet (2014, Research Diets) and tap water for all experiments except when noted.Adult male Sprague-Dawley rats (Charles River) weighing ~250-270 g (n=93) at arrival were housed under a 12-hour:12-hour light/dark cycle in a temperature-and humidity-controlled vivarium. Rats were individually housed in hanging wire-bottomed cage with ad libitum access to chow diet (Purina Lab Diet 5001), tap water and also had ad libitum access to kaolin pellets (Research Diets, K50001). Rats were exposed to kaolin for at least 5 days prior to measuring kaolin consumption in pica testing.Adult male shrews (Suncus murinus) weighing ~50-80 g (n=118 total), where bred and

The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of Peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist (GIPRA) reduced conditioned taste avoidance (CTA) without affecting hypophagia induced by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in the detection of aversive stimuli. Peripheral administration of a GIPRA induced neuronal activation (cFOS) in the AP. Further, whole-brain cFOS analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), an area of the brain that relays aversive/emetic stimuli to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPRA treatment reduces PYY-induced nausea-like behavior. Together, our study provides a novel mechanism by which GIP-based therapeutics may benefit the tolerability of weight loss agents.

BACKGROUND AND PURPOSE PA‐824 is a 2‐nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F420)‐dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des‐nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti‐mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA‐824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis.EXPERIMENTAL APPROACH PA‐824 (250 µM) was incubated with the 9000×g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis.KEY RESULTS PA‐824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des‐nitro product. Six of these products, but not the des‐nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn‐catalyzed des‐nitrification and radiolytic reduction of PA‐824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem‐catalyzed product of imidazole ring hydration (M2).CONCLUSIONS AND IMPLICATIONS PA‐824 was metabolized by des‐nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA‐824 was selectively bioactivated in M. tuberculosis and there was no evidence for ‘cross‐activation’ by human enzymes.

Glucagon-like peptide-1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by side effects including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Utilizing single-nuclei RNA-sequencing, we identified the cellular phenotypes of AP/NTS GIPR- and GLP-1R-expressing cells on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in GABAergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting.