LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic ß cells.

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning ß cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and ß cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human ß cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in ß cells to maintain appropriate insulin release.

GLP1R and GIPR expression and signaling in pancreatic alpha cells, beta cells and delta cells.

Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are transmembrane receptors involved in insulin, glucagon and somatostatin secretion from the pancreatic islet. Therapeutic targeting of GLP1R and GIPR restores blood glucose levels in part by influencing beta cell, alpha cell and delta cell function. Despite the importance of the incretin-mimetics for diabetes therapy, our understanding of GLP1R and GIPR expression patterns and signaling within the islet remain incomplete. Here, we present the evidence for GLP1R and GIPR expression in the major islet cell types, before addressing signaling pathway(s) engaged, as well as their influence on cell survival and function. While GLP1R is largely a beta cell-specific marker within the islet, GIPR is expressed in alpha cells, beta cells, and (possibly) delta cells. GLP1R and GIPR engage Gs-coupled pathways in most settings, although the exact outcome on hormone release depends on paracrine communication and promiscuous signaling. Biased agonism away from beta-arrestin is an emerging concept for improving therapeutic efficacy, and is also relevant for GLP1R/GIPR dual agonism. Lastly, dual agonists exert multiple effects on islet function through GIPR > GLP1R imbalance, increased GLP1R surface expression and cAMP signaling, as well as beneficial alpha cell-beta cell-delta cell crosstalk.

Synchronizing beta cells in the pancreas.

The secretion of insulin from the pancreas relies on both gap junctions and subpopulations of beta cells with specific intrinsic properties.

Fine-tuned photochromic sulfonylureas for optical control of beta cell Ca2+ fluxes.

We previously developed, synthesized and tested light-activated sulfonylureas for optical control of KATP channels and pancreatic beta cell activity in vitro and in vivo. Such technology relies on installation of azobenzene photoswitches onto the sulfonylurea backbone, affording light-dependent isomerization, alteration in ligand affinity for SUR1 and hence KATP channel conductance. Inspired by molecular dynamics simulations and to further improve photoswitching characteristics, we set out to develop a novel push-pull closed ring azobenzene unit, before installing this on the sulfonylurea glimepiride as a small molecule recipient. Three fine-tuned, light-activated sulfonylureas were synthesized, encompassing azetidine, pyrrolidine and piperidine closed rings. Azetidine-, pyrrolidine- and piperidine-based sulfonylureas all increased beta cell Ca2+ -spiking activity upon continuous blue light illumination, similarly to first generation JB253. Notably, the pyrrolidine-based sulfonylurea showed superior switch OFF performance to JB253. As such, third generation sulfonylureas afford more precise optical control over primary pancreatic beta cells, and showcase the potential of pyrrolidine-azobenzenes as chemical photoswitches across drug classes.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding.

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics' ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) - brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways.

PEPITEM modulates leukocyte trafficking to reduce obesity-induced inflammation.

Dysregulation of leukocyte trafficking, lipid metabolism, and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in lifestyle choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B cells were observed in the secondary lymphoid tissues (e.g. spleen and inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimize the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through lifestyle modifications.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Spatial distribution of heterogeneity as a modulator of collective dynamics in pancreatic beta-cell networks and beyond.

We study the impact of spatial distribution of heterogeneity on collective dynamics in gap-junction coupled beta-cell networks comprised on cells from two populations that differ in their intrinsic excitability. Initially, these populations are uniformly and randomly distributed throughout the networks. We develop and apply an iterative algorithm for perturbing the arrangement of the network such that cells from the same population are increasingly likely to be adjacent to one another. We find that the global input strength, or network drive, necessary to transition the network from a state of quiescence to a state of synchronised and oscillatory activity decreases as network sortedness increases. Moreover, for weak coupling, we find that regimes of partial synchronisation and wave propagation arise, which depend both on network drive and network sortedness. We then demonstrate the utility of this algorithm for studying the distribution of heterogeneity in general networks, for which we use Watts-Strogatz networks as a case study. This work highlights the importance of heterogeneity in node dynamics in establishing collective rhythms in complex, excitable networks and has implications for a wide range of real-world systems that exhibit such heterogeneity.

Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.

GC-Globulin/Vitamin D-Binding Protein Is Required for Pancreatic α-Cell Adaptation to Metabolic Stress.

GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in the transport of circulating vitamin 25(OH)D and fatty acids, as well as actin scavenging. In the pancreatic islets, the gene encoding GC, GC/Gc, is highly localized to glucagon-secreting α-cells. Despite this, the role of GC in α-cell function is poorly understood. We previously showed that GC is essential for α-cell morphology, electrical activity, and glucagon secretion. We now show that loss of GC exacerbates α-cell failure during metabolic stress. High-fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α-cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α-cell GC levels, increase glucagon granule area, and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α-cells of donors with type 2 diabetes, which is associated with changes in α-cell mass, morphology, and glucagon expression. Together, these data demonstrate an important role for GC in α-cell adaptation to metabolic stress.

Median eminence blood flow influences food intake by regulating ghrelin access to the metabolic brain.

Central integration of peripheral appetite-regulating signals ensures maintenance of energy homeostasis. Thus, plasticity of circulating molecule access to neuronal circuits involved in feeding behavior plays a key role in the adaptive response to metabolic changes. However, the mechanisms involved remain poorly understood despite their relevance for therapeutic development. Here, we investigated the role of median eminence mural cells, including smooth muscle cells and pericytes, in modulating gut hormone effects on orexigenic/anorexigenic circuits. We found that conditional activation of median eminence vascular cells impinged on local blood flow velocity and altered ghrelin-stimulated food intake by delaying ghrelin access to target neurons. Thus, activation of median eminence vascular cells modulates food intake in response to peripheral ghrelin by reducing local blood flow velocity and access to the metabolic brain.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Enzyme self-label-bound ATTO700 in single-molecule and super-resolution microscopy.

Herein, we evaluate near-infrared ATTO700 as an acceptor in SNAP- and Halo-tag protein labelling for Förster Resonance Energy Transfer (FRET) by ensemble and single molecule measurements. Microscopy of cell surface proteins in live cells is perfomed including super-resolution stimulated emission by depletion (STED) nanoscopy.

Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response.

Insulin-secreting ß-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder ß-cell state, critical for the islet first-phase response to glucose.

Metabolic Stress Impairs Pericyte Response to Optogenetic Stimulation in Pancreatic Islets.

Pancreatic islets are highly vascularized micro-organs ensuring whole body glucose homeostasis. Islet vascular cells play an integral part in sustaining adequate insulin release by beta cells. In particular, recent studies have demonstrated that islet pericytes regulate local blood flow velocity and are required for maintenance of beta cell maturity and function. In addition, increased metabolic demand accompanying obesity alters islet pericyte morphology. Here, we sought to explore the effects of metabolic stress on islet pericyte functional response to stimulation in a mouse model of type 2 diabetes, directly in the pancreas in vivo . We found that high fat diet induced islet pericyte hypertrophy without alterations in basal local blood flow. However, optogenetic stimulation of pericyte activity revealed impaired islet vascular responses, despite increased expression of genes encoding proteins directly or indirectly involved in cell contraction. These findings suggest that metabolic stress impinges upon islet pericyte function, which may contribute to beta cell failure during T2D.

Expanded LUXendin Color Palette for GLP1R Detection and Visualization In Vitro and In Vivo.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615, and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology.

Cell Networks in Endocrine/Neuroendocrine Gland Function.

Reproduction, growth, stress, and metabolism are determined by endocrine/neuroendocrine systems that regulate circulating hormone concentrations. All these systems generate rhythms and changes in hormone pulsatility observed in a variety of pathophysiological states. Thus, the output of endocrine/neuroendocrine systems must be regulated within a narrow window of effective hormone concentrations but must also maintain a capacity for plasticity to respond to changing physiological demands. Remarkably most endocrinologists still have a "textbook" view of endocrine gland organization which has emanated from 20th century histological studies on thin 2D tissue sections. However, 21st -century technological advances, including in-depth 3D imaging of specific cell types have vastly changed our knowledge. We now know that various levels of multicellular organization can be found across different glands, that organizational motifs can vary between species and can be modified to enhance or decrease hormonal release. This article focuses on how the organization of cells regulates hormone output using three endocrine/neuroendocrine glands that present different levels of organization and complexity: the adrenal medulla, with a single neuroendocrine cell type; the anterior pituitary, with multiple intermingled cell types; and the pancreas with multiple intermingled cell types organized into distinct functional units. We give an overview of recent methodologies that allow the study of the different components within endocrine systems, particularly their temporal and spatial relationships. We believe the emerging findings about network organization, and its impact on hormone secretion, are crucial to understanding how homeostatic regulation of endocrine axes is carried out within endocrine organs themselves. © 2022 American Physiological Society. Compr Physiol 12:3371-3415, 2022.

The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.