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.

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.

Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.

Fatty acid-binding protein 5 regulates diet-induced obesity via GIP secretion from enteroendocrine K cells in response to fat ingestion.

Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K cells in response to nutrient intake, especially fat. GIP is one of the contributing factors inducing fat accumulation that results in obesity. A recent study shows that fatty acid-binding protein 5 (FABP5) is expressed in murine K cells and is involved in fat-induced GIP secretion. We investigated the mechanism of fat-induced GIP secretion and the impact of FABP5-related GIP response on diet-induced obesity (DIO). Single oral administration of glucose and fat resulted in a 40% reduction of GIP response to fat but not to glucose in whole body FABP5-knockout (FABP5(-/-)) mice, with no change in K cell count or GIP content in K cells. In an ex vivo experiment using isolated upper small intestine, oleic acid induced only a slight increase in GIP release, which was markedly enhanced by coadministration of bile and oleic acid together with attenuated GIP response in the FABP5(-/-) sample. FABP5(-/-) mice exhibited a 24% reduction in body weight gain and body fat mass under a high-fat diet compared with wild-type (FABP5(+/+)) mice; the difference was not observed between GIP-GFP homozygous knock-in (GIP(gfp/gfp))-FABP5(+/+) mice and GIP(gfp/gfp)-FABP5(-/-) mice, in which GIP is genetically deleted. These results demonstrate that bile efficiently amplifies fat-induced GIP secretion and that FABP5 contributes to the development of DIO in a GIP-dependent manner.

Transcriptional regulatory factor X6 (Rfx6) increases gastric inhibitory polypeptide (GIP) expression in enteroendocrine K-cells and is involved in GIP hypersecretion in high fat diet-induced obesity.

Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K-cells in response to nutrient ingestion. GIP potentiates glucose-stimulated insulin secretion and induces energy accumulation into adipose tissue, resulting in obesity. Plasma GIP levels are reported to be increased in the obese state. However, the molecular mechanisms of GIP secretion and high fat diet (HFD)-induced GIP hypersecretion remain unclear, primarily due to difficulties in separating K-cells from other intestinal epithelial cells in vivo. In this study, GIP-GFP knock-in mice that enable us to visualize K-cells by enhanced GFP were established. Microarray analysis of isolated K-cells from these mice revealed that transcriptional regulatory factor X6 (Rfx6) is expressed exclusively in K-cells. In vitro experiments using the mouse intestinal cell line STC-1 showed that knockdown of Rfx6 decreased mRNA expression, cellular content, and secretion of GIP. Rfx6 bound to the region in the gip promoter that regulates gip promoter activity, and overexpression of Rfx6 increased GIP mRNA expression. HFD induced obesity and GIP hypersecretion in GIP-GFP heterozygous mice in vivo. Immunohistochemical and flow cytometry analysis showed no significant difference in K-cell number between control fat diet-fed (CFD) and HFD-fed mice. However, GIP content in the upper small intestine and GIP mRNA expression in K-cells were significantly increased in HFD-fed mice compared with those in CFD-fed mice. Furthermore, expression levels of Rfx6 mRNA were increased in K-cells of HFD-fed mice. These results suggest that Rfx6 increases GIP expression and content in K-cells and is involved in GIP hypersecretion in HFD-induced obesity.

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.

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.

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.

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.

Isoform-specific Roles of Prolyl Hydroxylases in the Regulation of Pancreatic ß-Cell Function.

Pancreatic ß-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated ß-cell-specific knockout (KO) mice for all 3 isoforms of PHD (ß-PHD1 KO, ß-PHD2 KO, and ß-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the ß-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for ß-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both ß-PHD1 KO and ß-PHD3 KO had elevated ß-cell apoptosis and reduced ß-cell mass. Isolated islets from ß-PHD1 KO and ß-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in ß-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, ß-PHD1 KO and ß-PHD3 KO mice had defective ß-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death.

Pancreatic ß-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8,is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of WT and global ZnT8-/- mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, WT islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNF-α, IFN-γ, IL1-ß) treatment induced islet cell death, but to a lesser extent in islets from ZnT8-/- mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in WT islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8-/- mice as compared to WT islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8-/- mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man.

Nicotinamide riboside has minimal impact on energy metabolism in mouse models of mild obesity.

Supplementation with precursors of NAD has been shown to prevent and reverse insulin resistance, mitochondrial dysfunction, and liver damage in mouse models of diet-induced obesity. We asked whether the beneficial effects of supplementation with the NAD precursor nicotinamide riboside (NR) are dependent on mouse strain. We compared the effects of NR supplementation on whole-body energy metabolism and mitochondrial function in mildly obese C57BL/6N and C57BL/6J mice, two commonly used strains to investigate metabolism. Male C57BL/6N and C57BL/6J mice were fed a high-fat diet (HFD) or standard chow with or without NR supplementation for 8 weeks. Body and organ weights, glucose tolerance, and metabolic parameters as well as mitochondrial O2 flux in liver and muscle fibers were assessed. We found that NR supplementation had no influence on body or organ weight, glucose metabolism or hepatic lipid accumulation, energy expenditure, or metabolic flexibility but increased mitochondrial respiration in soleus muscle in both mouse strains. Strain-dependent differences were detected for body and fat depot weight, fasting blood glucose, hepatic lipid accumulation, and energy expenditure. We conclude that, in mild obesity, NR supplementation does not alter metabolic phenotype in two commonly used laboratory mouse strains.

Prolyl-4-hydroxylase 3 maintains ß cell glucose metabolism during fatty acid excess in mice.

The α-ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting ß cells in vivo. Here, we show that the deletion of PHD3 specifically in ß cells (ßPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, ßPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in ßPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the ß cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

PDX1LOW MAFALOW ß-cells contribute to islet function and insulin release.

Transcriptionally mature and immature ß-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in ß-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH ß-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH ß-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the ß-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in ß-cell maturity, might be important for the maintenance of islet function.

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Persistent or Transient Human ß Cell Dysfunction Induced by Metabolic Stress: Specific Signatures and Shared Gene Expression with Type 2 Diabetes.

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived ß-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Informing ß-cell regeneration strategies using studies of heterogeneity.

BACKGROUND: Current therapeutic strategies for type 1 (T1DM) and type 2 diabetes mellitus (T2DM) rely on increasing or substituting endogenous insulin secretion in combination with lifestyle changes. ß-cell regeneration, a process whereby new ß-cells arise from progenitors, self-renewal or transdifferentiation, has the potential to become a viable route to insulin self-sufficiency. Current regeneration strategies capture many of the transcriptomic and protein features of native ß-cells, generating cells capable of glucose-dependent insulin secretion in vitro and alleviation of hyperglycemia in vivo. However, whether novel ß-cells display appreciable heterogeneity remains poorly understood, with potential consequences for long-term functional robustness. SCOPE OF REVIEW: The review brings together crucial discoveries in the ß-cell regeneration field with state-of-the-art knowledge regarding ß-cell heterogeneity. Aspects that might aid production of longer-lasting and more plastic regenerated ß-cells are highlighted and discussed. MAJOR CONCLUSIONS: Different ß-cell regeneration approaches result in a similar outcome: glucose-sensitive, insulin-positive cells that mimic the native ß-cell phenotype but which lack normal plasticity. The ß-cell subpopulations identified to date expand our understanding of ß-cell survival, proliferation and function, signposting the direction for future regeneration strategies. Therefore, regenerated ß-cells should exhibit stimulus-dependent differences in gene and protein expression, as well as establish a functional network with different ß-cells, all while coexisting with other cell types on a three-dimensional platform.

The role of beta cell heterogeneity in islet function and insulin release.

It is becoming increasingly apparent that not all insulin-secreting beta cells are equal. Subtle differences exist at the transcriptomic and protein expression levels, with repercussions for beta cell survival/proliferation, calcium signalling and insulin release. Notably, beta cell heterogeneity displays plasticity during development, metabolic stress and type 2 diabetes mellitus (T2DM). Thus, heterogeneity or lack thereof may be an important contributor to beta cell failure during T2DM in both rodents and humans. The present review will discuss the molecular and cellular features of beta cell heterogeneity at both the single-cell and islet level, explore how this influences islet function and insulin release and look into the alterations that may occur during obesity and T2DM.