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Internalisation of G protein-coupled receptors (GPCRs) can contribute to altered cellular responses by directing signalling from non-canonical locations, such as endosomes. If signalling processes are locally constrained, active receptors in different subcellular locations could produce different downstream effects. This phenomenon may be relevant to the optimal targeting of the glucagon-like peptide-1 receptor (GLP-1R), a type 2 diabetes and obesity target GPCR for which several ligands with varying internalisation tendency have been discovered. To investigate, we compared the signalling localisation effects of two prototypical GLP-1RAs with opposite signal bias and effects on GLP-1R trafficking: exendin-asp3 (ExD3), a full agonist that drives rapid internalisation, and exendin-phe1 (ExF1), which shows much slower internalisation. After using bioorthogonal labelling and fluorescent agonist conjugates to verify the divergent trafficking patterns of ExF1 and ExD3 in β-cell lines and primary pancreatic islets, we used live cell biosensors to monitor signalling at different subcellular locations. This revealed that cAMP/PKA/ERK signalling in β-cells is in fact distributed widely across the cell over short- (<5 min) and medium-term (up to 60 min) stimulation at pharmacological (>10 pM) concentrations, with no major differences in signal localisation that could be linked to internalised versus cell surface-bound GLP-1R. Moreover, washout experiments highlighted that, whilst fast-internalising ExD3 shows much greater accumulation and binding to GLP-1R in endosomes than slow-internalising ExF1, it is a rather inefficient driver of both cAMP production in β-cells and insulin secretion from perfused rat pancreata. These data provide a greater understanding of the cellular effects of biased GLP-1R agonism. Show less

Tirzepatide, a single-molecule dual glucose-dependent insulinotropic polypeptide (GIP)/glucagon-like peptide-1 (GLP-1) receptor (R) agonist, has shown superiority in the reduction of blood glucose and body weight, above selective GLP-1R agonists, but the contribution of GIP to these effects remains incompletely understood. To characterize the preclinical and in-human effects of a long-acting GIPR agonist monotherapy in healthy participants and patients with type 2 diabetes (T2D). A long-acting GIPR agonist (LY3537021) was characterized in vitro and in Long-Evans diet-induced obese rats and Wistar rats. Next, a phase 1, randomized, placebo-controlled, single ascending dose (SAD)/multiple ascending dose (MAD) study explored the safety, tolerability, pharmacokinetics, and pharmacodynamics of LY3537021 in healthy participants and participants with T2D in Singapore. In vitro, LY3537021 demonstrated potency greater than native GIP and selectivity for the GIPR. In vivo in rats, chronic treatment with LY3537021 resulted in weight loss and improved glycemic control during a glucose tolerance test. The phase 1 clinical study enrolled 85 healthy participants and patients with T2D (SAD, n = 47 [aged 25-64 years]; MAD, n = 38 [aged 25-69 years]; average baseline BMI was 25.9-27.0 kg/m In vivo studies demonstrated that LY3537021 reduced body weight and improved glycemia during a glucose challenge in rats. The phase 1 study demonstrated that the long-acting GIPR agonist LY3537021 was well tolerated, induced weight loss, and improved glucose control in humans. These observations better define the therapeutic benefit of long-acting GIPR agonists and support a distinct contribution of GIP agonism to the benefits observed with multi-agonist peptides that act via the GIPR. Future studies are needed in more diverse populations and in cohorts with overweight/obesity to confirm these findings. GOV: NCT04586907. Show less

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight. Show less

The incretin peptides glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptors coordinate β cell secretion that is proportional to nutrient intake. This effect permits consistent and restricted glucose excursions across a range of carbohydrate intake. The canonical signaling downstream of ligand-activated incretin receptors involves coupling to Gαs protein and generation of intracellular cAMP. However, recent reports have highlighted the importance of additional signaling nodes engaged by incretin receptors, including other G proteins and β-arrestin proteins. Here, the importance of Gαs signaling was tested in mice with conditional, postdevelopmental β cell deletion of Gnas (encoding Gαs) under physiological and pharmacological conditions. Deletion of Gαs/cAMP signaling induced immediate and profound hyperglycemia that responded minimally to incretin receptor agonists, a sulfonylurea, or bethanechol. While islet area and insulin content were not affected in Gnasβcell-/-, perifusion of isolated islets demonstrated impaired responses to glucose, incretins, acetylcholine, and IBMX In the absence of Gαs, incretin-stimulated insulin secretion was impaired but not absent, with some contribution from Gαq signaling. Collectively, these findings validate a central role for cAMP in mediating incretin signaling, but also demonstrate broad impairment of insulin secretion in the absence of Gαs that causes both fasting hyperglycemia and glucose intolerance. Show less

In combatting the obesity crisis, leveraging mechanisms that lower body weight is critical. The finding that treatment with tirzepatide, a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) receptor agonist, produces profound weight loss highlights the value of activating the incretin receptors. Supporting this, recent studies have revealed mechanisms by which GIP receptor (GIPR) activation is beneficial in pancreatic islets, the central nervous system (CNS), and adipose tissue. Paradoxically, a hypothesis has emerged that GIPR antagonism could be an additional option in treating obesity. This concept stems from concern that GIP facilitates lipid uptake and storage in adipose tissue, although the lipid-buffering capacity of adipocytes versus other cell types is metabolically favorable. In this article, we highlight the natural physiology of the incretins, noting GIP as the primary incretin. In the CNS, GIPR agonism attenuates nausea and suppresses appetite, features that also help GLP-1 receptor agonism promote a negative energy balance. Further, we provide rationale that, in protecting against ectopic fat distribution and augmenting substrate utilization to promote insulin sensitivity, GIPR activity in adipose tissue is advantageous. Collectively, these attributes support GIPR agonism in the treatment of obesity and metabolic disease. Show less

Agonists and antagonists of the glucose-dependent insulinotropic polypeptide receptor (GIPR) enhance body weight loss induced by glucagon-like peptide-1 receptor (GLP-1R) agonism. However, while GIPR agonism decreases body weight and food intake in a GLP-1R-independent manner via GABAergic GIPR Show less

Glucose-dependent insulinotropic polypeptide (GIP) was the first incretin identified and plays an essential role in the maintenance of glucose tolerance in healthy humans. Until recently GIP had not been developed as a therapeutic and thus has been overshadowed by the other incretin, glucagon-like peptide 1 (GLP-1), which is the basis for several successful drugs to treat diabetes and obesity. However, there has been a rekindling of interest in GIP biology in recent years, in great part due to pharmacology demonstrating that both GIPR agonism and antagonism may be beneficial in treating obesity and diabetes. This apparent paradox has reinvigorated the field, led to new lines of investigation, and deeper understanding of GIP. In this review, we provide a detailed overview on the multifaceted nature of GIP biology and discuss the therapeutic implications of GIPR signal modification on various diseases. Following its classification as an incretin hormone, GIP has emerged as a pleiotropic hormone with a variety of metabolic effects outside the endocrine pancreas. The numerous beneficial effects of GIPR signal modification render the peptide an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, drug-induced nausea and both bone and neurodegenerative disorders. Show less

Recent studies have found that glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism can enhance the metabolic efficacy of glucagon-like peptide-1 receptor agonist treatment by promoting both weight-dependent and -independent improvements on systemic insulin sensitivity. These findings have prompted new investigations aimed at better understanding the broad metabolic benefit of GIPR activation. Herein, we determined whether GIPR agonism favorably influenced the pharmacologic efficacy of the insulin-sensitizing thiazolidinedione (TZD) rosiglitazone in obese insulin-resistant (IR) mice. Genetic and pharmacological approaches were used to examine the role of GIPR signaling on rosiglitazone-induced weight gain, hyperphagia, and glycemic control. RNA sequencing was conducted to uncover potential mechanisms by which GIPR activation influences energy balance and insulin sensitivity. In line with previous findings, treatment with rosiglitazone induced the mRNA expression of the GIPR in white and brown fat. However, obese GIPR-null mice dosed with rosiglitazone had equivalent weight gain to that of wild-type (WT) animals. Strikingly, chronic treatment of obese IR WT animals with a long-acting GIPR agonist prevented rosiglitazone-induced weight-gain and hyperphagia, and it enhanced the insulin-sensitivity effect of this TZD. The systemic insulin sensitization was accompanied by increased glucose disposal in brown adipose tissue, which was underlined by the recruitment of metabolic and thermogenic genes. These findings suggest that GIPR agonism can counter the negative consequences of rosiglitazone treatment on body weight and adiposity, while improving its insulin-sensitizing efficacy at the same time. Show less

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943. Show less

Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT. To address this question, we utilized a murine model of diet-induced obesity and employed stable-isotope tracer studies in combination with metabolomic analyses in BAT and other tissues. Treatment with tirzepatide stimulated catabolism of BCAAs/BCKAs in BAT, as demonstrated by increased labeling of BCKA-derived metabolites, and increases in levels of byproducts of BCAA breakdown, including glutamate, alanine, and 3-hydroxyisobutyric acid (3-HIB). Further, chronic administration of tirzepatide increased levels of multiple amino acids in BAT that have previously been shown to be elevated in response to cold exposure. Finally, chronic treatment with tirzepatide led to a substantial increase in several TCA cycle intermediates (α-ketoglutarate, fumarate, and malate) in BAT. These findings suggest that tirzepatide induces a thermogenic-like amino acid profile in BAT, an effect that may account for reduced systemic levels of BCAAs in obese IR mice. Show less

A major challenge of obesity therapy is to sustain clinically relevant weight loss over time. Achieving this goal likely requires both reducing daily caloric intake and increasing caloric expenditure. Over the past decade, advances in pharmaceutical engineering of ligands targeting G protein-coupled receptors have led to the development of highly effective anorectic agents. These include mono-agonists of the GLP-1R and dual GIPR/GLP-1R co-agonists that have demonstrated substantial weight loss in experimental models and in humans. By contrast, currently, there are no medicines available that effectively augment metabolic rate to promote weight loss. Here, we present evidence indicating that activation of the GCGR may provide a solution to this unmet therapeutic need. In adult humans, GCGR agonism increases energy expenditure to a magnitude sufficient for inducing a negative energy balance. In preclinical studies, the glucagon-GCGR system affects key metabolically relevant organs (including the liver and white and brown adipose tissue) to boost whole-body thermogenic capacity and protect from obesity. Further, activation of the GCGR has been shown to augment both the magnitude and duration of weight loss that is achieved by either selective GLP-1R or dual GIPR/GLP-1R agonism in rodents. Based on the accumulation of such findings, we propose that the thermogenic activity of GCGR agonism will also complement other anti-obesity agents that lower body weight by suppressing appetite. Show less

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 (GIPR) agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist reduced conditioned taste avoidance (CTA) without affecting hypophagia mediated by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were found to be expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in detecting aversive stimuli. Peripheral administration of a GIPR agonist 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), a brainstem nucleus that relays aversive/emetic signals 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 GIPR agonist treatment reduces PYY-induced nausea-like behavior. Together, the results of our study indicate a novel mechanism by which GIP-based therapeutics may have benefit in improving the tolerability of weight loss agents. Show less

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist. Show less

During the past decade, pharmaceutical engineering of unimolecular agents has revealed the therapeutic potential of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism. From this work, one of the most intriguing findings is that engagement of GIPR enhances the weight loss profile of glucagon-like peptide 1 (GLP-1)-based therapeutics. Consequently, this pharmacological approach, in combination with novel Show less

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 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 whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT 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 of this, 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 acids (BCAAs) and ketoacids 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 and -independent 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. Show less

Glucose-dependent insulinotropic polypeptide (GIP) communicates nutrient intake from the gut to islets, enabling optimal levels of insulin secretion via the GIP receptor (GIPR) on β cells. The GIPR is also expressed in α cells, and GIP stimulates glucagon secretion; however, the role of this action in the postprandial state is unknown. Here, we demonstrate that GIP potentiates amino acid-stimulated glucagon secretion, documenting a similar nutrient-dependent action to that described in β cells. Moreover, we demonstrate that GIP activity in α cells contributes to insulin secretion by invoking paracrine α to β cell communication. Last, specific loss of GIPR activity in α cells prevents glucagon secretion in response to a meal stimulus, limiting insulin secretion and driving glucose intolerance. Together, these data uncover an important axis by which GIPR activity in α cells is necessary to coordinate the optimal level of both glucagon and insulin secretion to maintain postprandial homeostasis. Show less

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over β-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal β-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent. Show less

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in β-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces β-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Show less

Therapeutic engineering of glucagon-like peptide 1 (GLP-1) has enabled development of new medicines to treat type 2 diabetes. These injectable analogs achieve robust glycemic control by increasing concentrations of "GLP-1 equivalents" (∼50 pmol/L). Similar levels of endogenous GLP-1 occur after gastric bypass surgery, and mechanistic studies indicate glucose lowering by these procedures is driven by GLP-1. Therefore, because of the remarkable signaling and secretory capacity of the GLP-1 system, we sought to discover mechanisms that increase GLP-1 pharmacologically. To study active GLP-1, glucose-dependent insulinotropic polypeptide receptor ( Show less