Insulin and glucagon are described as having opposing actions on hepatic glycogen metabolism. However, here we showed that their coordinated action promoted glycogen turnover and meal glucose storage. In mice, pharmacological doses of insulin or glucagon failed to alter hepatic glycogen, but the combination produced a robust decrease in glycogen content. Additivity between insulin and glucagon was also seen with the activation of hepatic insulin signaling intermediates. This signaling pathway drove glycogen synthesis, suggesting concurrent actions on glycogen breakdown and repletion. A mixed nutrient meal, which stimulates an increase in both insulin and glucagon, enhanced the incorporation of dietary glucose into hepatic glycogen. This was much more pronounced than the effects of glucose alone, which only stimulated insulin secretion. These findings revealed that glucagon is required for efficient hepatic glucose storage when acting in concert with insulin. Coordinated insulin-glucagon signaling, thus, emerged as a critical mechanism for hepatic glycogen cycling, challenging the classical paradigm that these hormones work in opposition.

Introduction and Objective: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonism is foundational to obesity pharmacotherapies like semaglutide. These compounds were engineered for maximal G protein alpha(s) (Gsα) signaling potency and downstream cAMP production. However, this strategy requires reconsideration as partial, biased GLP-1R agonists characterized by decreased Gsα signaling and disproportionate reductions in β-arrestin recruitment relative to the native ligand provide greater weight loss than full, balanced agonists in preclinical models. Methods: We tested the hypothesis that in vitro signaling bias, which considers both cAMP signaling and β-arrestin recruitment, better predicts weight loss in diet induced obese (DIO) rodents than cAMP potency alone. Results: Our data demonstrates that the signaling bias metric β significantly correlates to GLP-1R agonist mediated weight loss in diet-induced obese mice. We further characterized a protracted GLP-1 analogue (NNC5840) which exhibits a partial, cAMP biased GLP-1R signaling profile in vitro and demonstrates superior maximal body weight reduction compared to semaglutide in DIO mice. The NNC5840 weight loss profile is characterized by reduced in vivo potency but increased maximal efficacy. Conclusion: These data suggest that drug discovery screening strategies which take a holistic approach to target receptor signaling may provide more efficacious candidate molecules. J.D. Douros: Employee; Novo Nordisk. D. Perez-Tilve: Research Support; MBX Biosciences, Novo Nordisk A/S. Other Relationship; BlueWater LLC, Ghrelco LLC. Consultant; Eli Lilly and Company, Ampeptec. M. Capozzi: None. B.N. DuBois: Employee; Novo Nordisk. R. Rohlfs: Employee; Novo Nordisk. J. Mokrosinski: Employee; Novo Nordisk. Stock/Shareholder; Novo Nordisk. R. Augustin: None. S.A. Mowery: Employee; Novo Nordisk. M. Waldhoer: Consultant; Schrödinger. P.J. Knerr: Employee; Novo Nordisk A/S. Consultant; Toralgen, Inc. This research was partially supported by Novo Nordisk and The European Union within the scope of the European Research Council ERC-CoG Trusted no.101044445, awarded to Timo D. Mueller.

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.

Introduction and Objective: Unimolecular triagonists drive substantial weight loss in patients with obesity (PwO) by engaging the glucagon-like peptide 1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) receptors to reduce food intake (FI) and the hepatic glucagon (Gcg) receptor to enhance energy expenditure (EE). However, their development has been challenged by deleterious cardiovascular (CV) effects including increased heart rate (HR), elongated QTc, and arrhythmia mediated by GcgR agonism. GLP-1R monoagonists on the other hand improve both obesity and CV outcomes with negligible effects on EE. We sought to imbue peptide GLP-1R agonists with an EE enhancing effect by combining them with ectopic GLP-1R expression and agonism in hepatocytes. Methods: We used an attenuated adenovirus (AAV) to induce the expression of a functional, liver-specific GLP-1R combined with traditional peptide agonist treatment to drive greater body weight loss via reduced energy intake and increased energy expenditure. Results: Agonism of the ectopic GLP-1R with either semaglutide, a low internalization GLP-1R agonist (Sema584), or a dual GLP-1R/GIPR agonist in wild-type (WT) diet induced obese (DIO) mice led to enhanced EE and improved weight loss compared to agonist treatment alone. Conclusion: This represents a novel mechanism for achieving polypharmacy to treat obesity. J.D. Douros: Employee; Novo Nordisk. M. Capozzi: None. J. Mokrosinski: Employee; Novo Nordisk. Stock/Shareholder; Novo Nordisk. B.N. DuBois: Employee; Novo Nordisk. R. Rohlfs: Employee; Novo Nordisk. S. Poulsen: Employee; Novo Nordisk. Stock/Shareholder; Novo Nordisk. E. Oude Blenke: None. K. Huus: Employee; Novo Nordisk A/S. S.A. Mowery: Employee; Novo Nordisk. D. D'Alessio: Consultant; Arrowhead Pharmaceuticals, Inc. Other Relationship; Eli Lilly and Company. Consultant; Gasherbrum Bio, Inc. Stock/Shareholder; MBX Biosciences. Consultant; Structure Therapeutics, Inc. Advisory Panel; Sun Pharmaceutical Industries Ltd. J. Campbell: Research Support; Eli Lilly and Company, Novo Nordisk. Advisory Panel; Structure Therapeutics, Inc. Research Support; Structure Therapeutics, Inc. Consultant; Arrowhead Pharmaceuticals, Inc. Advisory Panel; Boehringer-Ingelheim, Neurocrine, Roche Pharmaceuticals, Prostasis. Research Support; Merck & Co., Inc. P.J. Knerr: Employee; Novo Nordisk A/S. Consultant; Toralgen, Inc. D. Perez-Tilve: Research Support; MBX Biosciences, Novo Nordisk A/S. Other Relationship; BlueWater LLC, Ghrelco LLC. Consultant; Eli Lilly and Company, Ampeptec. This work was partially funded by Novo Nordisk. This work was partially funded by the European Union within the scope of the European Research Council ERC-CoG Trusted no. 101044445.

Many individuals with type 2 diabetes (T2D) cannot take current therapies due to their adverse effects. Thus, new glucose-lowering agents targeting unique mechanisms are needed. Studies have demonstrated that decreasing ketone oxidation, secondary to muscle-specific deletion of succinyl-CoA:3-ketoacid-CoA transferase (SCOT), protects mice against obesity-related hyperglycemia. In silico studies identified that the antipsychotic diphenylbutylpiperidines can inhibit SCOT and alleviate obesity-related hyperglycemia. Because ketones are a major brain fuel, whereas the diphenylbutylpiperidines have central nervous system-related adverse effects, we aimed to develop a peripheral selective SCOT inhibitor (PSSI). Using a pharmacophore derived from the diphenylbutylpiperidine-SCOT interaction, we synthesized PSSI-51, which inhibited SCOT activity in peripheral but not brain tissue, while decreasing myocardial ketone oxidation. Importantly, PSSI-51 treatment improved glycemia in obese mice and demonstrated reduced brain accumulation compared to the diphenylbutylpiperidine pimozide. We propose that PSSI-51 can lay the foundation for optimizing a new class of brain-impermeable SCOT inhibitors for treating T2D.

Many individuals with type 2 diabetes (T2D) cannot take current therapies due to their adverse effects. Thus, new glucose-lowering agents targeting unique mechanisms are needed. Studies have demonstrated that decreasing ketone oxidation, secondary to muscle-specific deletion of succinyl-CoA:3-ketoacid-CoA transferase (SCOT), protects mice against obesity-related hyperglycemia. In silico studies identified that the antipsychotic diphenylbutylpiperidines can inhibit SCOT and alleviate obesity-related hyperglycemia. Because ketones are a major brain fuel, whereas the diphenylbutylpiperidines have central nervous system-related adverse effects, we aimed to develop a peripheral selective SCOT inhibitor (PSSI). Using a pharmacophore derived from the diphenylbutylpiperidine-SCOT interaction, we synthesized PSSI-51, which inhibited SCOT activity in peripheral but not brain tissue, while decreasing myocardial ketone oxidation. Importantly, PSSI-51 treatment improved glycemia in obese mice and demonstrated reduced brain accumulation compared to the diphenylbutylpiperidine pimozide. We propose that PSSI-51 can lay the foundation for optimizing a new class of brain-impermeable SCOT inhibitors for treating T2D.

The advent of GPCR agonists and co-agonists that target the β-cell incretin receptors - GLP1R and GIPR - have revolutionized therapeutics for diabetes, though the signaling pathways downstream of these receptors have yet to be fully understood. Previous work in our lab has demonstrated the critical role of the glycolytic enzyme pyruvate kinase (PK) in the closure of KATP channels. However, PK activators also potentiate insulin secretion independent of KATP channel closure, suggesting additional roles for PK. We therefore sought to determine if PK potentiates insulin secretion downstream of GPCRs by modulating cAMP signaling. To elucidate the role of PK downstream of GPCRs, we employed live-cell imaging with subcellularly-targeted biosensors to track cAMP signaling over time in intact human and mouse islets in response to glucagon, GLP1, GIP, and tirzepatide. To further characterize the role of GLP1R and GCGR, chemical antagonism and mice deficient in β-cell GLP1R/GCGR were utilized in combination with TEPP-46, a pharmacologic activator of PKm2 and PKL. Our data demonstrates that PK amplifies cAMP signaling at the plasma membrane downstream of β-cell GPCRs. Specifically, there is enhanced protein kinase A (PKA) activity and increased insulin secretion in the presence of a PK activator with glucagon as a ligand. PK did not potentiate insulin secretion or PKA activity in response to GIP, indicating a receptor specific effect. Chemical antagonists and knockout experiments show that PK potentiates PKA activity primarily downstream GLP1R more than GCGR. These studies, which link glycolysis to GPCR signaling, suggest that biased GLP1R agonists that work via PK will more efficiently increase insulin secretion. Disclosure H.R. Foster: None. S.L. Lewandowski: None. R. Kirchner: None. M. Capozzi: None. J. Campbell: Research Support; Eli Lilly and Company, Novo Nordisk, Merck & Co., Inc. Advisory Panel; Structure Therapeutics, Inc. M.J. Merrins: None. Funding American Diabetes Association (1-18-JDF-017); NIH/NIDDK (R01DK113103); United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01BX005113); NIH/NIDDK (T32DK007665 and F31DK126403); HRSA (T32HP10010); NIH/NIA (T32AG000213); National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) (T32 DK007012; F32 DK116542); NIDDK, NIH (DK123075 and DK125353); United States Department of Veterans Affairs (I01 BX003700); NIH/NIDDK (R01 DK102598)

The fed state is often defined with a glucose-centric focus, where elevated glycemia stimulates insulin and reduces glucagon secretion. However, protein-containing meals that better reflect normal nutrition increase glucagon secretion, producing elevations in both hormones. Glucagon and insulin have opposing effects on hepatic glycogen; glucagon promotes glycogenolysis while insulin promotes storage. Yet, most work in this area has focused on the individual rather than combined actions of these hormones. The purpose of this study was to determine the effect of combined insulin and glucagon on postprandial hepatic glycogen metabolism. We hypothesized that their combined actions increase glycogen flux to enable storage of meal nutrients. Glucagon and/or insulin were injected into 5-hr fasted wild-type mice to determine the individual versus combined actions on hepatic glycogen content. Next, glucose alone versus a mixed-nutrient meal was gavaged with matching carbohydrate loads, using a 13C6-glucose tracer to assess incorporation of meal-derived glucose into glycogen. Finally, a mixed-nutrient meal with tracer was given to proglucagon null mice (Gcg-/-) to determine the effect on glycogen metabolism. Glucagon or insulin alone did not alter hepatic glycogen levels, however the combination of both hormones enhanced glycogenolysis. Meal-derived glucose incorporated into hepatic glycogen was higher after a mixed-nutrient meal compared to oral glucose alone. Gcg-/- mice stored significantly less meal-derived glucose as glycogen versus controls. Our results suggest that the combined actions of glucagon and insulin enhance hepatic glycogen turnover due to both enhanced glycogenolysis and storage of meal-derived glucose as glycogen. These actions enable the liver to have greater metabolic flexibility for nutrient storage in the postprandial state. This may have implications for disease states such as diabetes or metabolic-associated steatotic liver disease, where hormone action is altered. Disclosure M. Capozzi: None. D. Bouslov: None. A. Sargsyan: Employee; AstraZeneca. J. Campbell: Research Support; Eli Lilly and Company, Novo Nordisk, Merck & Co., Inc. Advisory Panel; Structure Therapeutics, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5K01DK129417)

Ocular levels of IL-1β, TNFα, IL-8, and IL-6 correlate with progression of diabetic retinopathy (DR). Müller cells (MC), which are crucial to maintaining retinal homeostasis, are targets and sources of these cytokines. We explored the relative capacities of these four DR-associated cytokines to amplify inflammatory signal expression both in and between human MC (hMC) and retinal microvascular endothelial cells (hRMEC) and in the mouse retina. Of the four cytokines, IL-1β was the most potent stimulus of transcriptomic alterations in hMC and hRMEC in vitro, as well as in the mouse retina after intravitreal injection in vivo. Stimulation with IL-1β significantly induced expression of all four transcripts in hMC and hRMEC. TNFα significantly induced expression of some, but not all, of the four transcripts in each cell, while neither IL-8 nor IL-6 showed significant induction in either cell. Similarly, conditioned media (CM) derived from hMC or hRMEC treated with IL-1β, but not TNFα, upregulated inflammatory cytokine transcripts in the reciprocal cell type. hRMEC responses to hMC-derived CM were dependent on IL-1R activation. In addition, we observed a correlation between cytokine expression changes following direct and CM stimulation and NFκB-p65 nuclear translocation in both hMC and hRMEC. Finally, in mice, intravitreal injections of IL-1β, but not TNFα, induced retinal expression of Il1b and CXCL8 homologues Cxcl1, Cxcl2, Cxcl3, and Cxcl5, encoding pro-angiogenic chemokines. Our results suggest that expression of IL-1β, TNFα, IL-8, and IL-6 may be initiated, propagated, and sustained by autocrine and paracrine signals in hRMEC and hMC through a process involving IL-1β and NFκB. Targeting these signals may help thwart inflammatory amplification, preventing progression to vision-threatening stages and preserving sight.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G proteincoupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, beta arrestin1 and beta arrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to beta arrestin is enhanced with signaling biased to a beta arrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitindependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGRK333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agoniststimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced beta arrestin1-dependent p38 signaling scaffold that requires cain health and disease.

Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. DR has non-proliferative stages, characterized in part by retinal neuroinflammation and ischemia, and proliferative stages, characterized by retinal angiogenesis. Several systemic factors, including poor glycemic control, hypertension, and hyperlipidemia, increase the risk of DR progression to vision-threatening stages. Identification of cellular or molecular targets in early DR events could allow more prompt interventions pre-empting DR progression to vision-threatening stages. Glia mediate homeostasis and repair. They contribute to immune surveillance and defense, cytokine and growth factor production and secretion, ion and neurotransmitter balance, neuroprotection, and, potentially, regeneration. Therefore, it is likely that glia orchestrate events throughout the development and progression of retinopathy. Understanding glial responses to products of diabetes-associated systemic dyshomeostasis may reveal novel insights into the pathophysiology of DR and guide the development of novel therapies for this potentially blinding condition. In this article, first, we review normal glial functions and their putative roles in the development of DR. We then describe glial transcriptome alterations in response to systemic circulating factors that are upregulated in patients with diabetes and diabetes-related comorbidities; namely glucose in hyperglycemia, angiotensin II in hypertension, and the free fatty acid palmitic acid in hyperlipidemia. Finally, we discuss potential benefits and challenges associated with studying glia as targets of DR therapeutic interventions. In vitro stimulation of glia with glucose, angiotensin II and palmitic acid suggests that: 1) astrocytes may be more responsive than other glia to these products of systemic dyshomeostasis; 2) the effects of hyperglycemia on glia are likely to be largely osmotic; 3) fatty acid accumulation may compound DR pathophysiology by promoting predominantly proinflammatory and proangiogenic transcriptional alterations of macro and microglia; and 4) cell-targeted therapies may offer safer and more effective avenues for DR treatment as they may circumvent the complication of pleiotropism in retinal cell responses. Although several molecules previously implicated in DR pathophysiology are validated in this review, some less explored molecules emerge as potential therapeutic targets. Whereas much is known regarding glial cell activation, future studies characterizing the role of glia in DR and how their activation is regulated and sustained (independently or as part of retinal cell networks) may help elucidate mechanisms of DR pathogenesis and identify novel drug targets for this blinding disease.

Mice systemically lacking dipeptidyl peptidase-4 (DPP4) have improved islet health, glucoregulation, and reduced obesity with high-fat diet (HFD) feeding compared to wild-type mice. Some, but not all, of this improvement can be linked to the loss of DPP4 in endothelial cells (ECs), pointing to the contribution of non-EC types. The importance of intra-islet signaling mediated by alpha to beta cell communication is becoming increasingly clear; thus, our objective was to determine if beta cell DPP4 regulates insulin secretion and glucose tolerance in HFD-fed mice by regulating the local concentrations of insulinotropic peptides. Using beta cell double incretin receptor knockout mice, beta cell- and pancreas-specific Dpp4(-/-) mice, we reveal that beta cell incretin receptors are necessary for DPP4 inhibitor effects. However, although beta cell DPP4 modestly contributes to high glucose (16.7 mM)-stimulated insulin secretion in isolated islets, it does not regulate whole-body glucose homeostasis.

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.

Pyruvate kinase (PK), by converting ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate, initiates insulin secretion via KATP closure and enhances insulin secretion via mitochondrial PEP production. The physiological drivers of mitochondrial PEP production include amino acids, which also work on neighboring α-cells to stimulate insulin secretion through activation of β-cell glucagon and GLP-1 receptors. We therefore hypothesized that PK would augment α-cell paracrine signaling upon amino acid (AA) stimulation.

The evolution of glucagon has seen the transition from an impurity in the preparation of insulin to the development of glucagon receptor agonists for use in type 1 diabetes. In type 2 diabetes, glucagon receptor antagonists have been explored to reduce glycemia thought to be induced by hyperglucagonemia. However, the catabolic actions of glucagon are currently being leveraged to target the rise in obesity that paralleled that of diabetes, bringing the pharmacology of glucagon full circle. During this evolution, the physiological importance of glucagon advanced beyond the control of hepatic glucose production, incorporating critical roles for glucagon to regulate both lipid and amino acid metabolism. Thus, it is unsurprising that the study of glucagon has left several paradoxes that make it difficult to distill this hormone down to a simplified action. Here, we describe the history of glucagon from the past to the present and suggest some direction to the future of this field.

Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1β, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1β-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1β-induced MC expression of IL-1β and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1β-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1β-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1β-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1β-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.

GIPR activity in α cells is required for the complete metabolic response to a meal.