Alpha Cells Research Articles

Pancreatic Islet Cells Response to IFNγ Relies on Their Spatial Location within an Islet

Type 1 diabetes (T1D) is an auto-immune disease characterized by the progressive destruction of insulin-producing pancreatic beta cells. While beta cells are the target of the immune attack, the other islet endocrine cells, namely the alpha and delta cells, can also be affected by the inflammatory milieu. Here, using a flow cytometry-based strategy, we compared the impact of IFNγ, one of the main cytokines involved in T1D, on the three endocrine cell subsets isolated from C57BL/6 mouse islets. RNA-seq analyses revealed that alpha and delta cells exposed in vitro to IFNγ display a transcriptomic profile very similar to that of beta cells, with an increased expression of inflammation key genes such as MHC class I molecules, the CXCL10 chemokine and the programmed death-ligand 1 (PD-L1), three hallmarks of IFNγ signaling. Interestingly, at low IFNγ concentration, we observed two beta cell populations (responders and non-responders) based on PD-L1 protein expression. Our data indicate that this differential sensitivity relies on the location of the cells within the islet rather than on the existence of two different beta cells subsets. The same findings were corroborated by the in vivo analysis of pancreatic islets from the non-obese diabetic mouse model of T1D, showing more intense PD-L1 staining on endocrine cells close to immune infiltrate. Collectively, our work demonstrates that alpha and delta cells are as sensitive as beta cells to IFNγ, and suggests a gradual diffusion of the cytokine into an islet. These observations provide novel insights into the in situ inflammatory processes occurring in T1D progression.

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD). Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression. A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease. The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

Single-cell transcriptomic and spatial landscapes of the developing human pancreas

Current differentiation protocols have not been successful in reproducibly generating fully functional human beta cells invitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single-cell RNA sequencing with spatial transcriptomics at multiple developmental time points and revealed distinct temporal-spatial gene cascades. Cell trajectory inference identified endocrine progenitor populations and branch-specific genes as the progenitors differentiate toward alpha or beta cells. Spatial differentiation trajectories indicated that Schwann cells are spatially co-located with endocrine progenitors, and cell-cell connectivity analysis predicted that they may interact via L1CAM-EPHB2 signaling. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for investigating human pancreas development for the research community.

Profiling RNA Editing in Single Cells.

RNA editing is a widespread molecular phenomenon occurring in a variety of organisms. In humans, it mainly involves the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs by ADAR enzymes. A-to-I RNA editing has been investigated in different tissues as well as in diverse experimental and pathological conditions. By contrast, its biological role in single cells has not been explored in depth. Recent methodologies for cell sorting in combination with deep sequencing technologies have enabled the study of eukaryotic transcriptomes at single cell resolution, paving the way to the profiling of their epitranscriptomic dynamics.Here we describe a step-by-step protocol to detect and characterize A-to-I events occurring in publicly available single-cell RNAseq experiments from human alpha and beta pancreatic cells.

Chemical Compounds and Ambient Factors Affecting Pancreatic Alpha-Cells Mass and Function: What Evidence?

The exposure to different substances present in the environment can affect the ability of the human body to maintain glucose homeostasis. Some review studies summarized the current evidence about the relationships between environment and insulin resistance or beta-cell dysfunction. Instead, no reviews focused on the relationships between the environment and the alpha cell, although in recent years clear indications have emerged for the pivotal role of the alpha cell in glucose regulation. Thus, the aim of this review was to analyze the studies about the effects of chemical, biological, and physical environmental factors on the alpha cell. Notably, we found studies focusing on the effects of different categories of compounds, including air pollutants, compounds of known toxicity present in common objects, pharmacological agents, and compounds possibly present in food, plus studies on the effects of physical factors (mainly heat exposure). However, the overall number of relevant studies was limited, especially when compared to studies related to the environment and insulin sensitivity or beta-cell function. In our opinion, this was likely due to the underestimation of the alpha-cell role in glucose homeostasis, but since such a role has recently emerged with increasing strength, we expect several new studies about the environment and alpha-cell in the near future.

Investigation of the effects of swimming exercises in rats given acrylamide

Acrylamide is a toxic substance used in industrial and laboratory processes. Acrylamide exposure has a toxic effect on many systems. Protective mechanisms should be developed against the effects caused by acrylamide. In our study, we investigated whether exercise has a protective effect against the changes that acrylamide will cause in pancreas. 32 adult Sprague-Dawley male rats were used. Control group was given only saline. Exercise group was applied swimming exercise for 1hour daily for 4 weeks. Acrylamide group was given 50mg/kg acrylamide by gavage for 4 weeks. Acrylamide+exercise group was applied 50mg/kg acrylamide for 4 weeks and swimming exercise for 1hour daily. After the experiment, fasting blood glucose and oral glucose tolerance test measurements were performed. Then, blood and pancreas samples were taken. Acrylamide exposure caused an increase in fasting blood glucose and oral glucose tolerance, a decrease in insulin levels and oxidative stress in acrylamide group. In exercise group, these values were similar to control group and no significant change was observed in acrylamide+exercise group. While there was an increase in the number of alpha cells in acrylamide group compared to the other groups, here was a decrease in the number of beta cells compared to control group. We can say that acrylamide causes changes in the islets of Langerhans by affecting alpha and beta cell numbers. The protective effect of exercise on beta and alpha cell mass was not statistically significant in the acrylamide+exercise group. When the results were examined, the decrease in oxidative stress and the higher number of beta and alpha cells in the acrylamide+exercise group compared to the acrylamide group suggested that 4 weeks of swimming exercise may have an effect on acrylamide exposure.

Restoring glutamate receptor signaling in pancreatic alpha cells rescues glucagon responses in type 1 diabetes

Glucagon secretion from pancreatic alpha cells is crucial to prevent hypoglycemia. People with type 1 diabetes lose this glucoregulatory mechanism and are susceptible to dangerous hypoglycemia for reasons still unclear. Here we determine that alpha cells in living pancreas slices from donors with type 1 diabetes do not mount an adequate glucagon response and cannot activate the positive autocrine feedback mediated by AMPA/kainate glutamate receptors. This feedback is required to elicit full glucagon responses in the healthy state. Reactivating residual AMPA/kainate receptor function with positive allosteric modulators restores glucagon secretion in human slices from donors with type 1 diabetes as well as glucose counterregulation in non-obese diabetic mice. Our study thus identifies a defect in autocrine signaling that contributes to alpha cell failure. The use of positive allosteric modulators of AMPA/kainate receptors overcomes this deficiency and prevents hypoglycemia, an effect that could be used to improve the management of diabetes.

Preclinical exploration of combined glucagon inhibition and liver-preferential insulin for treatment of diabetes using in vitro assays and rat and mouse models

Aims/hypothesisNormalisation of blood glucose in individuals with diabetes is recommended to reduce development of diabetic complications. However, risk of severe hypoglycaemia with intensive insulin therapy is a major obstacle that prevents many individuals with diabetes from obtaining the recommended reduction in HbA1c. Inhibition of glucagon receptor signalling and liver-preferential insulin action have been shown individually to have beneficial effects in preclinical models and individuals with diabetes (i.e. improved glycaemic control), but also have effects that are potential safety risks (i.e. alpha cell hyperplasia in response to glucagon receptor antagonists and increased levels of liver triacylglycerols and plasma alanine aminotransferase activity in response to glucagon receptor antagonists and liver-preferential insulin). We hypothesised that a combination of glucagon inhibition and liver-preferential insulin action in a dual-acting molecule would widen the therapeutic window. By correcting two pathogenic mechanisms (dysregulated glucagon signalling and non-physiological distribution of conventional insulin administered s.c.), we hypothesised that lower doses of each component would be required to obtain sufficient reduction of hyperglycaemia, and that the undesirable effects that have previously been observed for monotreatment with glucagon antagonists and liver-preferential insulin could be avoided.MethodsA dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule was designed and tested in rodent models (normal rats, rats with streptozotocin-induced hyperglycaemia, db/db mice and mice with diet-induced obesity and streptozotocin-induced hyperglycaemia), allowing detailed characterisation of the pharmacokinetic and pharmacodynamic properties of the dual-acting molecule and relevant control compounds, as well as exploration of how the dual-acting molecule influenced glucagon-induced recovery and spontaneous recovery from acute hypoglycaemia.ResultsThis molecule normalised blood glucose in diabetic models, and was markedly less prone to induce hypoglycaemia than conventional insulin treatment (approximately 4.6-fold less potent under hypoglycaemic conditions than under normoglycaemic conditions). However, compared to treatment with conventional long-acting insulin, this dual-acting molecule also increased triacylglycerol levels in the liver (approximately 60%), plasma alanine aminotransferase levels (approximately twofold) and alpha cell mass (approximately twofold).Conclusions/interpretationWhile the dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule showed markedly improved regulation of blood glucose, effects that are potential safety concerns persisted in the pharmacologically relevant dose range.Graphical abstract

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Targeted delivery of harmine to xenografted human pancreatic islets promotes robust cell proliferation

Type 1 diabetes (T1D) occurs as a consequence of the autoimmune destruction of insulin-producing pancreatic beta (β) cells and commonly presents with insulin deficiency and unregulated glycemic control. Despite improvements in the medical management of T1D, life-threatening complications are still common. Beta-cell replication to replace lost cells may be achieved by using small-molecule mitogenic drugs, like harmine. However, the safe and effective delivery of such drugs to beta cells remains a challenge. This work aims to deploy an antibody conjugated nanocarrier platform to achieve cell-specific delivery of candidate therapeutic and imaging agents to pancreatic endocrine cells. We approached this goal by generating core–shell type micellar nanocarriers composed of the tri-block copolymer, Pluronic®F127 (PEO100–PPO65–PEO100). We decorated these nanocarriers with a pancreatic endocrine cell-selective monoclonal antibody (HPi1), with preference for beta cells, to achieve active targeting. The PPO-based hydrophobic core allows encapsulation of various hydrophobic cargoes, whereas the PEO-based hydrophilic shell curbs the protein adhesion, hence prolonging the nanocarriers' systemic circulation time. The nancarriers were loaded with quantum dots (QDots) that allowed nanocarrier detection both in-vitro and in-vivo. In-vitro studies revealed that HPi1 conjugated nanocarriers could target endocrine cells in dispersed islet cell preparations with a high degree of specificity, with beta cells exhibiting a fluorescent quantum dot signal that was approximately five orders of magnitude greater than the signal associated with alpha cells. In vivo endocrine cell targeting studies demonstrated that the HPi1 conjugated nanocarriers could significantly accumulate at the islet xenograft site. For drug delivery studies, the nanocarriers were loaded with harmine. We demonstrated that HPi1 conjugated nanocarriers successfully targeted and delivered harmine to human endocrine cells in a human islet xenograft model. In this model, targeted harmine delivery yielded an ~ 41-fold increase in the number of BrdU positive cells in the human islet xenograft than that observed in untreated control mice. By contrast, non-targeted harmine yielded an ~ 9-fold increase in BrdU positive cells. We conclude that the nanocarrier platform enabled cell-selective targeting of xenografted human pancreatic endocrine cells and the selective delivery of the hydrophobic drug harmine to those cells. Further, the dramatic increase in proliferation with targeted harmine, a likely consequence of achieving higher local drug concentrations, supports the concept that targeted drug delivery may promote more potent biological responses when using harmine and/or other drugs than non-targeting approaches. These results suggest that this targeted drug delivery platform may apply in drug screening, beta cell regenerative therapies, and/or diagnostic imaging in patients with type 1 diabetes.

Pancreatic alpha cell glucagon–liver FGF21 axis regulates beta cell regeneration in a mouse model of type 2 diabetes

Aims/hypothesisGlucagon receptor (GCGR) antagonism ameliorates hyperglycaemia and promotes beta cell regeneration in mouse models of type 2 diabetes. However, the underlying mechanisms remain unclear. The present study aimed to investigate the mechanism of beta cell regeneration induced by GCGR antagonism in mice.MethodsThe db/db mice and high-fat diet (HFD)+streptozotocin (STZ)-induced mice with type 2 diabetes were treated with antagonistic GCGR monoclonal antibody (mAb), and the metabolic variables and islet cell quantification were evaluated. Plasma cytokine array and liver RNA sequencing data were used to screen possible mediators, including fibroblast growth factor 21 (FGF21). ELISA, quantitative RT-PCR and western blot were applied to verify FGF21 change. Blockage of FGF21 signalling by FGF21-neutralising antibody (nAb) was used to clarify whether FGF21 was involved in the effects of GCGR mAb on the expression of beta cell identity-related genes under plasma-conditional culture and hepatocyte co-culture conditions. FGF21 nAb-treated db/db mice, systemic Fgf21-knockout (Fgf21−/−) diabetic mice and hepatocyte-specific Fgf21-knockout (Fgf21Hep−/−) diabetic mice were used to reveal the involvement of FGF21 in beta cell regeneration. A BrdU tracing study was used to analyse beta cell proliferation in diabetic mice treated with GCGR mAb.ResultsGCGR mAb treatment improved blood glucose control, and increased islet number (db/db 1.6±0.1 vs 0.8±0.1 per mm2, p<0.001; HFD+STZ 1.2±0.1 vs 0.5±0.1 per mm2, p<0.01) and area (db/db 2.5±0.2 vs 1.2±0.2%, p<0.001; HFD+STZ 1.0±0.1 vs 0.3±0.1%, p<0.01) in diabetic mice. The plasma cytokine array and liver RNA sequencing data showed that FGF21 levels in plasma and liver were upregulated by GCGR antagonism. The GCGR mAb induced upregulation of plasma FGF21 levels (db/db 661.5±40.0 vs 466.2±55.7 pg/ml, p<0.05; HFD+STZ 877.0±106.8 vs 445.5±54.0 pg/ml, p<0.05) and the liver levels of Fgf21 mRNA (db/db 3.2±0.5 vs 1.8±0.1, p<0.05; HFD+STZ 2.0±0.3 vs 1.0±0.2, p<0.05) and protein (db/db 2.0±0.2 vs 1.4±0.1, p<0.05; HFD+STZ 1.6±0.1 vs 1.0±0.1, p<0.01). Exposure to plasma or hepatocytes from the GCGR mAb-treated mice upregulated the mRNA levels of characteristic genes associated with beta cell identity in cultured mouse islets and a beta cell line, and blockage of FGF21 activity by an FGF21 nAb diminished this upregulation. Notably, the effects of increased beta cell number induced by GCGR mAb were attenuated in FGF21 nAb-treated db/db mice, Fgf21−/− diabetic mice and Fgf21Hep−/− diabetic mice. Moreover, GCGR mAb treatment enhanced beta cell proliferation in the two groups of diabetic mice, and this effect was weakened in Fgf21−/− and Fgf21Hep−/− mice.Conclusions/interpretationOur findings demonstrate that liver-derived FGF21 is involved in the GCGR antagonism-induced beta cell regeneration in a mouse model of type 2 diabetes.Graphical abstract

RF04 | PSUN135 Genetic Inactivation of Glutaminase Reduces Amino Acid Induced Alpha Cell Proliferation

Abstract Glucagon stimulates glycogenolysis and gluconeogenesis in liver resulting in increased hepatic glucose output. Thus, interrupting glucagon signaling in liver reduces blood glucose levels in animal models and individuals with diabetes. However, interrupting glucagon signaling also leads to hyperaminoacidemia and proliferation of glucagon secreting alpha cells, evidence of an endocrine liver-alpha cell axis. Among the high amino acids, we previously identified high glutamine levels are required for alpha cell proliferation. We recently found that glutaminase (GLS) is expressed in alpha cells at much higher levels than any other pancreatic endocrine cells. Additionally, mouse pancreatic islets cultured in high amino acid containing media and treated with the small molecule inhibitor of GLS (CB839) have reduced alpha cell proliferation (Vehicle: 7.6 ± 2.2%, 0.1µM CB839: 0.11 ± 0.05%**, 10µM: 0.02 ± 0.01%**; n=3-4, **p &amp;lt; 0.01). To further characterize the role of glutamine metabolism in glutamine-induced alpha cell proliferation, GLS activity was specifically knocked out in mouse alpha cells by breeding Gcg-CRE ERT2 with Glsflox mice (aGlsKO). Targeted deletion of Gls expression in most alpha cells was confirmed by staining for GLS and glucagon (aGlsWT: 95% GLS+, aGlsKO: 18% GLS+). Mice were treated with a glucagon receptor monoclonal antibody (GCGR mAb/mAb-4) for 10 days or with isotype control IgG. aGlsWT mice treated with GCGR mAb have robust alpha cell proliferation while aGlsKO mice treated with GCGR mAb for 10 days showed a 4.5-fold decrease in alpha cell proliferation and were similar to mice treated with the control IgG (aGlsWT Ab: 15.7 ± 3.6% alpha cell proliferation, aGlsWT IgG: 1.0 ± 0.4%***, aGlsKO Ab: 3.4 ± 0.6%**; n=4-6, ***p &amp;lt; 0.001). In addition, aGlsWT mice treated with GCGR mAb showed a robust activation of mTOR in alpha cells as measured by phospho-S6 expression while this was lost in aGlsKO mice treated with GCGR mAb. Induced expression of the mTOR-dependent glutamine transporter SLC38A5 was also lost (aGlsWT Ab: 25.0 ± 4.8% alpha cell SLC38A5 expression, aGlsWT IgG: 0.40 ± 0.27%***, aGlsKO Ab: 3.7 ± 3.5%***, n= 2-4, ***p &amp;lt; 0.001). Similar results on proliferation and mTOR activation in alpha cells were observed after 4 weeks of GCGR mAb treatment. Together these data suggest a critical role for glutamine metabolism via glutaminase in alpha cell proliferation. Presentation: Saturday, June 11, 2022 1:36 p.m. - 1:41 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

LBSUN331 Rescuing Alpha Cell Responses To Hypoglycemia In Type 1 Diabetes

Abstract Increased glucagon secretion from the pancreatic alpha cell is the first and most important defense against hypoglycemia. In type 1 diabetes this defensive mechanism is lost, increasing the mortality risk. In addition to the reduced glucagon response to hypoglycemia in T1D, basal glucagon secretion is elevated further stimulating hyperglycemia and desensitizing autocrine signaling pathways. Furthermore, alpha cells lose their ability to respond appropriately to rising glucose levels. In healthy individuals, rising glucose levels lead to increased insulin secretion, resulting in inhibition of glucagon secretion. In T1D however, the loss of beta cells results in the absence of these paracrine signals that mediate glucagon suppression under hyperglycemic conditions. We tested the hypothesis that reactivating paracrine and autocrine signaling pathways pharmacologically reverses alpha cell glucose blindness. We used living pancreas slices, which allow functional assessments of damaged and infiltrated islets within their native environment. We studied tissues from non-diabetic donors and donors with type 1 diabetes to determine alpha cell responses to (a) changes in glycemia, (b) agonists, antagonists, and positive allosteric modulators of autocrine and paracrine signaling pathways, and (c) reference stimuli such as adrenaline and KCl depolarization using functional recordings. We further performed in vivo studies using mouse models with defective glucose counterregulation to determine whether alpha cell responses to hypoglycemia could be restored. We found that human alpha cells in slices from type 1 diabetic donors had normal glucagon content and responded to KCl depolarization but failed to respond to decreases in glucose concentration. Furthermore, we found severely diminished Ca2+ responses to both lowering in glucose concentration and glutamate receptor stimulation. Our in vitro results not only reproduced important features seen in vivo but also revealed mechanistic defects related to autocrine and paracrine inputs. Importantly, reestablishing these inputs pharmacologically rescued glucagon secretion in response to a lowering in glucose concentration in human tissue slices/islets from donors with T1D as well as to hypoglycemia in the nonobese diabetic (NOD) mouse model. Several of these signaling molecules are used in other medical indications and could be repurposed to develop therapies aimed at preventing hypoglycemia in T1D. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

PSUN134 Loss of Glp-1r Signaling in the β-Cell Impairs Adaptive Proliferation and Insulin Secretion

Abstract Type 2 diabetes mellitus is characterized by insulin resistance and loss of pancreatic b-cell mass. b-cells are located in the pancreatic islets and secrete insulin. Though many therapies exist that address insulin resistance and to augment b-cell insulin secretion, there is a critical need to find therapies to preserve b-cell mass in order to decrease the incidence and severity of diabetes. In non-diabetic obesity, an early adaptive response to insulin resistance is increased b-cell proliferation. This compensatory mechanism leads to increased b-cell mass and increased insulin secretion from b-cells. This increase in b-cell mass is seen in as little as 4 days of high fat diet (HFD) feeding in murine models. A promising therapeutic for the preservation of b-cell mass is glucagon-like peptide-1 receptor (Glp-1r) agonists. The ability of GLP-1 to stimulate b-cell proliferation and inhibit apoptosis is largely based on studies using pharmacologic treatment, but the importance of GLP-1 in b-cell mass regulation in normal physiology or pathophysiology has not been well studied. Notably, GLP-1 is secreted from alpha cells in the local islet environment and this paracrine signaling pathway is important for islet function. The goal of this work is to investigate the contribution of b-cell Glp-1r signaling to b-cell mass regulation in the metabolic stress condition of a one-week HFD. We hypothesize that b-cell Glp-1r signaling is necessary for the compensatory mechanisms needed to maintain glucose homeostasis during metabolic stress conditions and that lack of b-cell Glp-1r will lead to decreased proliferation of b-cells. Understanding the role of β-cell Glp-1r signaling in adaptive b-cell mass expansion will allow for development of new strategies to augment β-cell mass in type 2 diabetes. We used a newly generated murine model with a b-cell specific knockout of Glp-1r, where the Ins1-Cre knock-in transgene drives recombination in b-cells. Glp-1r fl/fl mice (WT) and Glp-1r fl/fl – Ins1Cre mice (KO) were fed a HFD for one week. There was a trend toward elevated fasting (171+/-29 mg/dl vs 205+/-36 mg/dl, p=0.0475, n=9) and non-fasting blood glucose (195+/-24 mg/dl vs 216+/-40 mg/dl, p=0.0537, n=18) and impaired glucose tolerance in the KO mice. Notably, we found that insulin secretion in response to intraperitoneal glucose is impaired in KO mice. KO mice have a blunted proliferation response to a 1-week HFD stress, as measured by Ki67 mRNA levels (1.5-fold induction of Ki67 in WT vs. 0.6-fold induction in KO, p=0.0167, n=8-10). b-cell proliferation will also be measured by immunofluorescent image analysis of whole pancreas sections. These data suggest there is a critical role for b-cell Glp-1r signaling in the early proliferative response of b-cells in response to metabolic stress. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Investigating Alpha and Beta Cell Phenotypes in Type 1 Diabetes

In type 1 diabetes (T1D), β-cells are destroyed by autoimmunity, resulting in lifelong dependencies on insulin therapy. There is considerable heterogeneity in the pathology, genetics and response to therapies in T1D, however, little research has focused on how T1D α-cells and surviving β-cells behave. Our previous work studying type 2 diabetes showed that α-cell impairment is heterogeneous. Here, we apply a similar rationale to investigate behaviours of α- and β-cells in T1D.

LBSUN289 Ketonuria — A Possible Early Indicator Of Impending Euglycemic DKA In Setting Of Recent SGLT-2 Inhibitor Use And Acute Stress

Abstract Introduction - Euglycemic DKA is a notorious entity; previously thought to be primarily afflicting type 1 diabetics and rarely type 2 diabetics in a state of relative insulin deficiency, it has now become a major cause of concern and lead to the issuance of FDA warning in 2015 with regards to its link with SGLT-2 inhibitors. Although DKA is characterized by hyperglycemia, metabolic acidosis and ketogenesis, euglycemic DKA poses a diagnostic challenge as it remains disguised under the cover of euglycemia. Moreover, in the setting of recent SGLT-2 inhibitor use, this definition becomes even more of a paradox as SGLT-2 inhibitors, by the virtue of their action, promote ketogenesis thereby further adding to this enigma. Case presentation - A 67 year old male with hypertension and diabetes mellitus type 2 (on Metformin and Empagliflozin) presented to the emergency room with dizziness. He woke up to go to the bathroom that day and felt weak, dizzy and like he was going to fall. He endorsed nausea and vomiting but denied headache, fever, chills, chest pain, shortness of breath, abdominal pain, and problems with urination/bowel movements. He was afebrile and hemodynamically stable. On examination, left sided dysmetria, loss of coordination, and axial imbalance were noted. No motor/sensory deficits. Labs were significant for WBC count 10 k/mm cu (4 - 11), Creatinine 0.78 mg/dL (0.7 - 1.3), Glucose 279 mg/dL (70 - 99), Bicarbonate 21 mmol/L (21 -31), Anion gap 19 mmol/L (6 - 14), and Lactate 3.2 mmol/L (0.7 - 2). Urinalysis showed glucosuria (glucose &amp;gt;500 mg/dL) and ketonuria (ketones 20 mg/dl). CT head was negative but MRI brain showed left inferior cerebellar peduncle acute ischemic infarct. Stroke protocol was initiated and he transferred to telemetry. The next day, labs showed Glucose 166, Bicarbonate 13, Anion gap 21, and Ketones 5.6 mmol/L (&amp;lt;0.6). Since blood glucose levels remained less than 250 mg/dl with concurrent anion gap metabolic acidosis in the setting of recent Empagliflozin use, he was transferred to ICU for insulin infusion for management of euglycemic diabetic ketoacidosis. Subsequently, he was transitioned to a basal-bolus insulin regimen and transferred to the floors. Discussion - SGLT-2 inhibitors reduce renal tubular glucose reabsorption, causing glycosuria, and promote ketonemia directly by enhancing ketogenesis via glucagon secretion from the pancreatic alpha cells and indirectly by decreasing the renal clearance of ketone bodies. In the setting of acute stress, such as stroke in our patient, and recent SGLT-2 inhibitor use, the presence of ketonuria should be recognized as an early ominous warning sign for euglycemic DKA. A targeted approach detailing diagnostic and therapeutic interventions should be designed to halt its progression in its track before it transforms into the dreaded euglycemic DKA. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Association between osteocalcin, a pivotal marker of bone metabolism, and secretory function of islet beta cells and alpha cells in Chinese patients with type 2 diabetes mellitus: an observational study

BackgroundSeveral recent studies have found that Osteocalcin (OCN), a multifunctional protein secreted exclusively by osteoblasts, is beneficial to glucose metabolism and type 2 diabetes mellitus (T2DM). However, the effects of OCN on islets function especially islet ɑ cells function in patients with type 2 diabetes mellitus characterized by a bi-hormonal disease are still unclear. The purpose of this cross-sectional study was to investigate the relationship between serum OCN and the secretion of islet β cells and ɑ cells in Chinese patients with type 2 diabetes mellitus.Methods204 patients with T2DM were enrolled. Blood glucose (FBG, PBG0.5h, PBG1h, PBG2h, PBG3h), insulin (FINS, INS0.5h, INS1h, INS2h, INS3h), C-peptide (FCP, CP0.5h, CP1h, CP2h, CP3h), and glucagon (GLA0, GLA0.5 h, GLA1h, GLA2h, GLA3h) levels were measured on 0 h, 0.5 h, 1 h, 2 h, and 3 h after a 100 g standard bread meal load. Early postprandial secretion function of islet β cells was calculated as Δcp0.5h = CP0.5-FCP. The patients were divided into low, medium and high groups (T1, T2 and T3) according to tertiles of OCN. Comparison of parameters among three groups was studied. Correlation analysis confirmed the relationship between OCN and pancreatic secretion. Multiple regression analysis showed independent contributors to pancreatic secretion.Main resultsFBG, and PBG2h were the lowest while Δcp0.5h was the highest in the highest tertile group (respectively, p < 0.05). INS3h, area under the curve of insulin (AUCins3h) in T3 Group were significantly lower than T1 Group (respectively, p < 0.05). GLA1h in T3 group was lower than T1 group (p < 0.05), and GLA0.5 h in T3 group was lower than T2 and T1 groups (p < 0.05). Correlation analysis showed OCN was inversely correlated with Homeostatic model of insulin resistance (HOMA-IR), INS3h, AUCins3h (p < 0.05), and was still inversely correlated with FCP, GLA0.5 h, GLA1h, area under the curve of glucagon (AUCgla3h) (respectively, p < 0.05) after adjustment for body mass index (BMI) and alanine aminotransferase (ALT). The multiple regression analysis showed that OCN was independent contributor to Δcp0.5h, GLA0.5h and GLA1h (respectively, p < 0.05).ConclusionsHigher serum OCN level is closely related to better blood glucose control, higher insulin sensitivity, increased early-phase insulin secretion of islet β cells and appropriate inhibition of postprandial glucagon secretion of islet ɑ cells in adult patients with type 2 diabetes mellitus.

Analysis of an ABO subtype with a novel variation

To analyze the molecular characteristics of a ABO subgroup. The ABO phenotype was determined with the tube method. Exons of the ABO gene were analyzed by Sanger sequencing, and haplotypes of exons 6 and 7 were analyzed by cloning sequencing. By forward typing, the red blood cells showed 3+ agglutination reaction with anti-A and 4+ agglutination with anti-B. A weak reaction with A1 cells and no agglutination reaction with B, O cells by the reverse typing. Sequencing results showed heterozygosity including c.297A>G, c.467C>T, c.526C>G, c.608A>G, c.657C>T, c.703G>A, c.796C>A, c.803G>C, c.930G>A. Cloning sequencing revealed a c.608A>G variant in the A allele compared with the ABO*A1.02. A new variant site of subtype A of c.608G variation has been identified.

Alpha cell dysfunction in type 1 diabetes is independent of a senescence program.

Type 1 Diabetes (T1D) is caused by insulin deficiency, due to progressive autoimmune destruction of pancreatic β cells. Glucagon-secreting α cells become dysfunctional in T1D and contribute to pathophysiology, however, the mechanisms involved are unclear. While the majority of β cells are destroyed in T1D, some β cells escape this fate and become senescent but whether α cell dysfunction involves a senescence program has not been explored. Here we addressed the question of whether α cells become senescent during the natural history of T1D in the non-obese diabetic (NOD) mouse model and humans. NOD mice had several distinct subpopulations of α cells, but none were defined by markers of senescence at the transcriptional or protein level. Similarly, α cells of human T1D donors did not express senescence markers. Despite the lack of senescence in α cells in vivo, using a human islet culture model, we observed that DNA damage-induced senescence led to alterations in islet glucagon secretion, which could be rescued by inhibiting the senescence-associated secretory phenotype (SASP). Together our results suggest that α cell dysfunction in T1D is not due to activation of a senescence program, however, senescent β cell accumulation in the islet microenvironment may have a negative effect on α cell function.

Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells

The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.