Pancreatic Alpha Cells Research Articles

1465-P: Metabolic Remodeling in Pancreatic Beta Cells in Japanese Patients with Type 2 Diabetes

Background and Aims: It has been shown that metabolic remodeling from oxidative phosphorylation to glycolysis via activation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) occurs in pancreatic beta cells in non-Asians with diabetes, resulting in impaired beta cell function. We used multiple autopsy pancreatic sections to investigate whether such changes occur in Japanese patients with type 2 diabetes (T2D) , and if so, which clinical and pathological features of islets relate to this remodeling. Methods: Autopsy pancreatic sections from patients with T2D and non-diabetic donors (ND) were stained with antibodies against PFKFB3, insulin, and glucagon. The PFKFB3-positive ratio of pancreatic beta cells and alpha cells between T2D and ND were compared. Next, the relationships between PFKFB3-postive beta cell ratio, patient background factors and pathological features of the islets were explored. Results: In total, 36 cases (18 cases each for T2D and ND) were enrolled and 50-100 islets/case were evaluated. Mean age was 70.6 ± 7.7 vs. 64.3 ± 12.8 years and body mass index (BMI) 22.4 ± 4.2 vs. 23.3 ± 4.5 kg/m2, respectively. T2D duration was 9.8 ± 9.9 years. The PFKFB3-positive beta cell ratio was significantly higher in T2D (29.7 ± 18.6% vs. 11.1 ± 8.7%, p<0.05) , whereas the PFKFB3-positive alpha cell ratio did not differ between groups. A negative correlation between PFKFB3-positive beta cell ratio and BMI was found only in the T2D group (r=-0.65, p<0.05) . No correlation was observed between PFKFB3-positive beta cell ratio and age/disease duration in the T2D group. Thioflavin T staining revealed a positive correlation between amyloid deposition in the islets of T2D and PFKFB3 positivity in beta cells (r=0.67, p<0.05) . Conclusion: Our results indicate that intracellular metabolic remodeling mediated by upregulation of PFKFB3 occurs in pancreatic beta cells of Japanese patients with T2D. Low BMI and amyloid deposition may affect PFKFB3 expression in beta cells from patients with T2D. Disclosure R.Izumihara: None. H.Nomoto: None. K.Chiba: Research Support; Grant for Young Researcher from Japan Association for Diabetes Education and Care, Japan Diabetes Society Junior Scientist Development Grant, Japan Society for the Promotion of Science , MSD Life Science Foundation, Promotion for Young Research Talent and Network, The Akiyama Life Science Foundation, The Uehara Memorial Foundation. H.Kameda: None. K.Cho: None. A.Nakamura: Research Support; Kissei Pharmaceutical Co., Ltd., MSD, Nippon Boehringer Ingelheim, Taisho Pharmaceutical Holdings Co., Ltd. H.Miyoshi: Research Support; Abbott Japan Co., Ltd., Boehringer Ingelheim International GmbH, Daiichi Sankyo, Kowa Company, Ltd., LifeScan, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Astellas Pharma Inc., Boehringer Ingelheim International GmbH, Eli Lilly and Company, Kowa Company, Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd. H.Mizukami: None. T.Atsumi: Consultant; AbbVie Inc., AstraZeneca, MEDICAL & BIOLOGICAL LABORATORIES CO., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Ono Pharmaceutical Co., Ltd., Pfizer Inc., Research Support; AbbVie Inc., Alexion Pharmaceuticals, Inc., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Speaker's Bureau; AbbVie Inc., Astellas Pharma Inc., Bristol-Myers Squibb Company, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo, Eisai Co., Ltd., Eli Lilly and Company, Kyowa Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Pfizer Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, UCB, Inc. Funding JSPS KAKENHI Grant Number JP20281167.Japan Diabetes Society Junior Scientist Development Grant.MSD Life Science Foundation, Public Interest Incorporated Foundation.

Necrolytic migratory erythema is an important visual cutaneous clue of glucagonoma

Glucagonoma is an extremely rare neuroendocrine tumor that arises from pancreatic islet alpha cells. Although glucagonoma is usually accompanied by a variety of characteristic clinical symptoms, early diagnosis is still difficult due to the scarcity of the disease. In this study, we present the cumulative experiences, clinical characteristics and treatments of seven patients diagnosed with glucagonoma during the past 10 years at the First Affiliated Hospital of Xi’an Jiaotong University. The seven patients in our cohort consisted of six females and one male with an average diagnosis age of 40.1 years (range 23–51). The average time from onset of symptoms to diagnosis of glucagonoma was 14 months (range 2–36 months). All the patients visited dermatology first for necrolytic migratory erythema (NME) 7/7 (100%), and other presenting symptoms included diabetes mellitus (DM) 4/7 (57%), stomatitis 2/7 (28%), weight loss 4/7 (57%), anemia 4/7 (57%), diarrhea 1/7 (14%), and DVT1/7 (14%). Plasma glucagon levels were increased in all patients (range 216.92–3155 pg/mL) and declined after surgery. Imaging studies revealed that four of seven patients had liver metastasis. Six of seven patients received surgical resection, and all of them received somatostatin analog therapy. Symptoms improved significantly in 6 out of 7 patients. Three of seven patients died of this disease by the time of follow-up. Our data suggest that if persistent NME is associated with DM and high glucagon levels, timely abdominal imaging should be performed to confirm glucagonoma. Once diagnosed, surgery and somatostatin analogs are effective for symptom relief and tumor control.

Urotensin II receptor knockout alleviates streptozotocin‐induced hyperglucagonemia and diabetic kidney disease

Plasma and urinary levels of UII, a potent vasoactive peptide, are elevated in diabetes mellitus (DM) patients. UII receptor (UT) expression levels are also increased in the kidneys of humans and animals with DM. Palosuran, an orally active UT antagonist, increased insulin levels and improved renal function in uninephrectomized streptozotocin (STZ)‐treated rats. However, human studies on the potential use of palosuran for kidney protection in diabetes were inconclusive. In addition to UT antagonism, palosuran can activate somatostatin receptors. Also, the reduced affinity of palosuran for UT in intact cells and tissues suggests that it may not be an optimal UT antagonist. Thus, using non‐selective and less potent UT antagonists constitutes a weakness in the scientific rigor of the prior attempts to dissect UT as a potential therapeutic target in DKD. Hence, studies using genetic animal models and highly selective and potent pharmacological antagonists are needed to fill the gaps of understanding on the pathophysiological significance of the UII system in DM. In the present study, we examine the development of hyperglycemia and DKD in the wild‐type (WT) and UT knockout (KO) mouse model of type 1 DM.STZ‐treated mice showed increased blood glucose levels, glucosuria, decreased plasma insulin levels, and diabetic kidney disease (DKD), which UT KO attenuated. However, UT KO failed to reverse STZ‐induced insulin deficiency. To understand the underlying mechanism for alleviating hyperglycemia and DKD in UT KO despite the uncorrected insulin deficiency, we tested the role of the primary insulin counterregulatory hormone, glucagon. We found that STZ treatment increased plasma glucagon levels in WT mice but not in UT KO mice. This finding was supported by double immunohistochemistry of pancreatic islet sections, where STZ treatment reduced insulin and increased glucagon staining. UT KO reversed only the effect of STZ on glucagon staining. These findings suggest that UT KO protects the glucagon‐producing alpha cells but not the insulin‐producing beta cells.UII increased glucagon secretion in a cultured pancreatic alpha cell line, which UT inhibition reversed. Next, to determine how UII triggers glucagon secretion, we performed membrane potential recordings in alpha cells using the perforated‐patch clamp technique. UII produced depolarization of alpha cells. This effect was abolished by UT inhibition. Calcium (Ca2+) imaging demonstrated that UII stimulates intracellular Ca2+ ([Ca2+]i) elevation in alpha cells, which T‐ and L‐type Ca2+ channel blockers attenuated.Taken together, the results of this study suggest that: (1) STZ‐treatment causes insulin deficiency, hyperglycemia, glucosuria, DKD, and hyperglucagonemia. (2) UT KO reverses STZ effects, except insulin deficiency. (3) UII increases glucagon secretion by alpha cells, mediated by UII‐induced membrane depolarization and Ca2+ influx via T‐ and L‐type Ca2+ channels. Therefore, the reversal of hyperglycemia, glucosuria, and DKD in UT KO might be linked to the reversal of hyperglucagonemia.

Modeling the Amino Acid Effect on Glucagon Secretion from Pancreatic Alpha Cells.

Type 2 Diabetes Mellitus (T2DM) is a burdensome problem in modern society, and intensive research is focused on better understanding the underlying cellular mechanisms of hormone secretion for blood glucose regulation. T2DM is a bi-hormonal disease, and in addition to 100 years of increasing knowledge about the importance of insulin, the second hormone glucagon, secreted by pancreatic alpha cells, is becoming increasingly important. We have developed a mathematical model for glucagon secretion that incorporates all major metabolic processes of glucose, fatty acids, and glutamine as the most abundant postprandial amino acid in blood. In addition, we consider cAMP signaling in alpha cells. The model predictions quantitatively estimate the relative importance of specific metabolic and signaling pathways and particularly emphasize the important role of glutamine in promoting glucagon secretion, which is in good agreement with known experimental data.

Loss of urotensin II receptor diminishes hyperglycemia and kidney injury in streptozotocin-treated mice.

Beyond the CNS, urotensin II (UII) and its receptor (UT) are functionally expressed in peripheral tissues of the endocrine, cardiovascular, and renal systems. The expression levels of UII and UT in the kidney and circulating UII levels are increased in diabetes. UII also promotes mesangial proliferation and matrix accumulation in vitro. Here, we evaluate the effect of UT deletion on the development of hyperglycemia and diabetic kidney disease (DKD) in streptozotocin (STZ)-treated mice. Ten-week-old WT and UT knockout (KO) mice were injected with STZ for 5 days to induce diabetes. Blood glucose levels were measured weekly, and necropsy was performed 12 weeks after STZ injection. UT ablation slowed hyperglycemia and glucosuria in STZ-treated mice. UT KO also ameliorated STZ-induced increase in HbA1c, but not STZ-induced decrease in plasma insulin levels. However, STZ-induced increases in plasma glucagon concentration and immunohistochemical staining for glucagon in pancreatic islets were lessened in UT KO mice. UT ablation also protected against STZ-induced kidney derangements, including albuminuria, mesangial expansion, glomerular lesions, and glomerular endoplasmic reticulum stress. UT is expressed in a cultured pancreatic alpha cell line, and its activation by UII triggered membrane depolarization, T- and L-type voltage-gated Ca2+channel-dependent Ca2+influx, and glucagon secretion. These findings suggest that apart from direct action on the kidneys to cause injury, UT activation by UII may result in DKD by promoting hyperglycemia via induction of glucagon secretion by pancreatic alpha cells.

Acetyl-CoA-carboxylase 1 (ACC1) plays a critical role in glucagon secretion

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

A glycolytic enzyme complex and creatine kinase locally and differentially regulate ATP-sensitive potassium (KATP) channels in pancreatic islet alpha and beta cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. Here, using the inside-out excised patch configuration, we isolated the plasma membrane compartment containing KATP on β-cells and α-cells from humans and mice. The characteristic response of KATP to inhibition by ATP and activation by ADP was used to identify the presence of a glycolytic enzyme complex that is functionally coupled to KATP regulation. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in β-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally pyruvate kinase.

Artemether and aspterric acid induce pancreatic alpha cells to transdifferentiate into beta cells in zebrafish.

Recently, the antimalarial drug, artemether and the neurotransmitter GABA were identified to convert alpha cells into beta-like cells in vivo. However, some of these observations were challenged by other studies. To help address the controversy, we took advantage of zebrafish as a model to perform this study. First, we performed a small-molecule screening for artemether and its skeleton analogues. Second, we used the Cre-LoxP system for lineage tracing to indicate the conversion of alpha cells into beta cells in vivo. The stable transgenic ins2:eGFP αTC1-6-cell line was used for evaluation of alpha-cell transdifferentiation in vitro. We further used multiple zebrafish transgenic and mutation lines to demonstrate beta-cell differentiation, beta-cell ablation and alpha-cell hyperplasia in this study. We showed that artemether and another sesquiterpene, aspterric acid, induced alpha-cell transdifferentiation into beta cells, both in zebrafish as well as using αTC1-6 cells. Furthermore, these two compounds also converted alpha cells into beta cells when beta cells were lost or alpha cells were hyperplastic in zebrafish. Unlike the previous report, the conversion of alpha cells to beta cells was mediated by increasing Pax4 expression, but not suppression of Arx expression. Our data suggest that in zebrafish and αTC1-6 cells, both artemether and aspterric acid induce alpha-cell transdifferentiation. Our data, along with those of Li et al. (2017), suggested that artemether and aspterric acid were able to induce alpha-cell transdifferentiation, at least in zebrafish and αTC1-6 cells.

On the Electrophysiological Component of Pancreatic Alpha-Cell Models.

Glucagon, the main hormone responsible for increasing blood glucose levels, is secreted by pancreatic alphacells in a Ca2+ dependent process associated to membrane potential oscillations developed by the dynamic operation of K+, Na+ and Ca2+ channels. The mechanisms behind membrane potential and Ca2+ oscillations in alpha-cells are still under debate, and some new research works have used alpha-cell models to describe electrical activity. In this paper we studied the dynamics of electrical activity of three alpha-cell models using the Lead Potential Analysis method and Bifurcation Diagrams. Our aim is to highlight the differences in their dynamic behavior and therefore, in their response to glucose. Both issues are relevant to understand the stimulus-secretion coupling in alpha-cells and then, the mechanisms behind their dysregulation in Type 2 Diabetes.Clinical Relevance - A reliable description of the electrophysiological mechanisms in pancreatic alpha-cells is key to understand and treat the dysregulation of these cells in patients with Type 2 Diabetes.

Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia.

Patients with diabetes mellitus exhibit hyperglucagonemia, or excess glucagon secretion, which may be the underlying cause of the hyperglycemia of diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may drive the development of hyperglucagonemia. Therefore, uncovering the mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus.

The Role of Pancreatic Alpha Cells and Endothelial Cells in the Reduction of Oxidative Stress in Pseudoislets

Pancreatic beta cells have inadequate levels of antioxidant enzymes, and the damage induced by oxidative stress poses a challenge for their use in a therapy for patients with type 1 diabetes. It is known that the interaction of the pancreatic endocrine cells with support cells can improve their survival and lead to less vulnerability to oxidative stress. Here we investigated alpha (alpha TC-1), beta (INS1E) and endothelial (HUVEC) cells assembled into aggregates known as pseudoislets as a model of the pancreatic islets of Langerhans. We hypothesised that the coculture of alpha, beta and endothelial cells would be protective against oxidative stress. First, we showed that adding endothelial cells decreased the percentage of oxidative stress-positive cells. We then asked if the number of endothelial cells or the size (number of cells) of the pseudoislet could increase the protection against oxidative stress. However, no additional benefit was observed by those changes. On the other hand, we identified a potential supportive effect of the alpha cells in reducing oxidative stress in beta and endothelial cells. We were able to link this to the incretin glucagon-like peptide-1 (GLP-1) by showing that the absence of alpha cells in the pseudoislet caused increased oxidative stress, but the addition of GLP-1 could restore this. Together, these results provide important insights into the roles of alpha and endothelial cells in protecting against oxidative stress.

A Metabolomic Signature of Glucagon Action in Healthy Individuals With Overweight/Obesity.

ContextGlucagon is produced and released from the pancreatic alpha-cell to regulate glucose levels during periods of fasting. The main target for glucagon action is the liver, where it activates gluconeogenesis and glycogen breakdown; however, glucagon is postulated to have other roles within the body.ObjectiveWe sought to identify the circulating metabolites that would serve as markers of glucagon action in humans.MethodsIn this study (NCT03139305), we performed a continuous 72-hour glucagon infusion in healthy individuals with overweight/obesity. Participants were randomized to receive glucagon 12.5 ng/kg/min (GCG 12.5), glucagon 25 ng/kg/min (GCG 25), or a placebo control. A comprehensive metabolomics analysis was then performed from plasma isolated at several time points during the infusion to identify markers of glucagon activity.ResultsGlucagon (GCG 12.5 and GCG 25) resulted in significant changes in the plasma metabolome as soon as 4 hours following infusion. Pathways involved in amino acid metabolism were among the most affected. Rapid and sustained reduction of a broad panel of amino acids was observed. Additionally, time-dependent changes in free fatty acids and diacylglycerol and triglyceride species were observed.ConclusionThese results define a distinct signature of glucagon action that is broader than the known changes in glucose levels. In particular, the robust changes in amino acid levels may prove useful to monitor changes induced by glucagon in the context of additional glucagon-like peptide-1 or gastric inhibitory polypeptide treatment, as these agents also elicit changes in glucose levels.

1192-P: Robust Arginine-Stimulated Glucagon Secretion in Patients with Type 1 Diabetes Independent of Diabetes Duration, Age, HbA1c, and Beta-Cell Secretory Capacity

In type 1 diabetes (T1D), the counterregulatory glucagon response to hypoglycemia disappears over time. It remains unknown whether this is due to lack of glucagon production or represents a malfunctioning stimulus-secretion coupling in pancreatic alpha cells. Arginine test (5 g of arginine infused intravenously (iv) over 1 minute after an overnight fast) with frequent measurements of plasma glucagon and serum C-peptide concentrations over 30 minutes was performed in 80 patients with T1D (19/61 females/males, age 49 (22-74) years (median (range)), duration 20 (2-46) years, BMI 28 (22-44) kg/m2, HbA1c 8.2 (7.5-10) %). The interrelationship between the arginine-induced glucagon response and demographic, clinical and biochemical parameters was evaluated using a stepwise approach in two multiple linear regression analysis models with glucagon area under curve (AUC) and Cmax, respectively, as dependent parameter. Mean baseline plasma glucagon concentrations amounted to 10.6±5.5 pmol/L (mean±SD). Overall, robust arginine-induced glucagon responses were observed (AUC: 499±209 pmol/L × min; Cmax: 41.0±17.7 pmol/L) independently of diabetes duration, age, HbA1c and beta cell secretory capacity. Fasting plasma glucagon and waist-to-hip ratio predicted the glucagon response in both models. When leaving out fasting plasma glucagon of the models, fasting low-density lipoprotein cholesterol and alanine aminotransferase as well as eGFR also predicted the glucagon response. In conclusion, glucagon secretory capacity in patients with T1D as assessed by iv arginine test seems preserved independently of diabetes duration, age, HbA1c and beta cell secretory capacity positioning pharmaceutical restoration of the glucagon counterregulatory response to hypoglycemia as an attractive therapeutic strategy for the protection of insulin-induced hypoglycemia. Disclosure J. Warnøe: None. T. F. Dejgaard: Advisory Panel; Self; Novo Nordisk, Consultant; Self; Boehringer Ingelheim International GmbH, Research Support; Self; AstraZeneca, Novo Nordisk, Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk. F. K. Knop: Advisory Panel; Self; MSD Corporation, Novo Nordisk A/S, Sanofi, Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Novo Nordisk A/S, Pharmacosmos, Zealand Pharma A/S, Research Support; Self; Novo Nordisk A/S, Zealand Pharma A/S, Speaker's Bureau; Self; AstraZeneca, Bayer AG, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, MSD Corporation, Novo Nordisk A/S. N. J. Johansen: Employee; Self; Novo Nordisk. A. Lund: Speaker's Bureau; Self; Novo Nordisk, Sanofi. J. I. Bagger: None. N. J. Wewer albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Regeneron Pharmaceuticals Inc., Speaker's Bureau; Self; Merck & Co., Inc., Mercodia. B. Hartmann: None. J. J. Holst: Consultant; Self; Novo Nordisk, Other Relationship; Self; Antag Therapeutics, Bainan Biotech, MSD Corporation, Novo Nordisk, Other Relationship; Spouse/Partner; Antag Therapeutics, Bainan Biotech, Synklino ApS. T. Vilsbøll: Consultant; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Gilead Sciences, Inc., Lilly Diabetes, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk, Sanofi, Sun Pharmaceutical Industries Ltd. H. U. Andersen: Stock/Shareholder; Self; Novo Nordisk A/S.

Prognostic transcriptome classes of duodenopancreatic neuroendocrine tumors.

Duodenopancreatic neuroendocrine tumors (DPNETs) aggressiveness is heterogeneous. Tumor grade and extension are commonly used for prognostic determination. Yet, grade classes are empirically defined, with regular updates changing the definition of classes. Genomic screening may provide more objective classes and reflect tumor biology. The aim of this study was to provide a transcriptome classification of DPNETs. We included 66 DPNETs, covering the entire clinical spectrum of the disease in terms of secretion, grade, and stage. Three distinct molecular groups were identified, associated with distinct outcomes (log-rank P < 0.01): (i) better-outcome DPNETs with pancreatic beta-cell signature. This group was mainly composed of well-differentiated, grade 1 insulinomas; (ii) poor-outcome DPNETs with pancreatic alpha-cell and hepatic signature. This group included all neuroendocrine carcinomas and grade 3 DPNETs, but also some grade 1 and grade 2 DPNETs and (iii) intermediate-outcome DPNETs with pancreatic exocrine and progenitor signature. This group included grade 1 and grade 2 DPNETs, with some insulinomas. Fibrinogen gene FGA expression was one of the topmost expressed liver genes. FGA expression was associated with disease-free survival (HR = 1.13, P = 0.005) and could be validated on two independent cohorts. This original pathophysiologic insight provides new prognostic classification perspectives.

Functional Heterogeneity Among Pancreatic Alpha Cells

Historically, endocrine cells in the pancreatic islets have been assumed to function as relatively homogenous populations largely because we lacked the ability to measure individual cell activity with sufficient throughput to reliably detect heterogeneity within each population. The glucagon-secreting alpha cells play a vital role in regulating glycemia, but the mechanisms that control alpha cell activity and whether the alpha cells behave as a single unit or heterogeneously remain incompletely understood. To overcome the limitations in throughput that have to date prevented the study of alpha cells at the population level, we used genetically-encoded fluorescent indicators selectively expressed in alpha cells. Imaging intact mouse islets with these indicators in 3D responding to treatments in real time yields hundreds of individual alpha cell recordings per experiment. Calcium imaging showed reproducible heterogeneous responses to a panel of known physiological potentiators of glucagon secretion such as arginine vasopressin, epinephrine, and amino acids. Separate dose response experiments revealed that the proportion of alpha cells responding to each signal plateaus at different proportions of alpha cells. The calcium data correlate both with direct glucagon secretion levels as well as cAMP measurement. Our findings highlight previously unappreciated levels of functional heterogeneity among alpha cells and demonstrate that alpha cells are not a single uniform unit. Our observations suggest that dose-dependent increases in glucagon secretion in response to different physiological cues may be the result of mobilizing progressively larger proportions of the total alpha cell mass. We hypothesize that this functional heterogeneity is a built-in mechanism through which different physiological cues elicit graded glucagon responses from the alpha cells.

Cerebral Venous Sinus Thrombosis as a First Presentation of Glucagonoma

Background: Glucagonoma is a very rare type of pancreatic neuroendocrine tumor (pNET) which represent about 2% of all pNETs (Yao et al.). Although glucagonoma can occur in multiple endocrine tumor syndromes (e.g., MEN-1), most cases are non-hereditary (John & Schwartz). The typical 4Ds presentation are diabetes (DM), depression, dermatosis, & deep vein thrombosis (DVT) (Cunha-Silva et al.). About 50% of glucagonoma patients have DVT during their disease course (Feingold et al.). Most reported DVT cases in glucagonoma are either lower limb DVT or pulmonary embolism (Teixeira, Nico & Ghideti; Castro et al.). To our knowledge, there is no report of a cerebral venous sinus thrombosis (CVST) as the first presentation for glucagonoma. Clinical Case: A 67-year-old male with a history of psoriasis & benign prostatic hyperplasia presented to the emergency department with a diffuse headache. He was sent home with conservative management. Four days later, he developed acute left-sided weakness with numbness & facial droop. CT head with contrast revealed a superior sagittal sinus thrombosis extending into the right transverse & right sigmoid sinuses. Heparin infusion was initiated. Due to the extensive sinus vein thrombosis & lack predisposing conditions, a pan CT was done for possible malignancy. The scan showed a solid, well-defined, markedly enhancing lesion in the pancreatic tail, suggestive of a NET. At the same time, a new diagnosis of type 2 DM was made based on HbA1C of 11.8%. The patient denied any diarrhea, fever, abdominal pain or episodes suggestive of hypoglycemia. However, he endorsed night sweats with weight loss. His family history was significant for a brother who had a pNET that was resected. Endocrinology was consulted, & a workup for pNET was ordered. Chromogranin A (CgA) was elevated at 439.9 ng/ml (<=82), while on PPI. 24-hour urine cortisol was normal. PTH, serum calcium, & the pituitary hormonal panel were all normal. hCG & CEA were normal. Given the new onset DM & DVT plasma glucagon was requested & came back elevated at 202 pg/ml (reference range 80 pg/ml). MRI showed an hypervascular T2 isointense lesion at the pancreas’ tail measuring 10 x 11 x 10 mm. A Ga68-DOTATATE PET scan was ordered. With this constellation of findings, we initiated somatostatin analog therapy while surgical resection was planned. We referred the patient to medical genetics. Conclusion: DVT is a common presentation in glucagonoma & a major cause of mortality. The possible mechanism appears to be related to increased secretion of factor X by pancreatic alpha cells (Lobo et al.). Although DVT is not unusual, an extensive CVST as the first presentation in glucagonoma has not been reported. In this case, & in addition to unexplained DVT, the new diagnosis of DM in an elderly patient raised the suspicion of secondary causes. Such a presentation should encourage clinicians to broaden the differential diagnoses, including pNET.

Patient-Specific Risk Factors and Clinical Correlates of Euglycemic Diabetic Ketoacidosis in Patients on Sodium-Glucose Co-Transporter-2 Inhibitors

Background: Sodium-Glucose Co-Transporter-2 Inhibitors (SGLT-2i) association with euglycemic diabetic ketoacidosis (EDKA) has been well reported. The underlying mechanism is mainly enhanced lipolysis and ketone bodies’ reabsorption. They also stimulate the pancreatic alpha cells and inhibit the beta cells, thereby causing an imbalance in glucagon/insulin levels, further contributing to lipolysis and ketogenesis. SGLT-2i were also found to cause EDKA in all types of diabetes, even uncovering undiagnosed Latent Autoimmune Diabetes of the Adult (LADA). Methods: Numerous electronic databases were systematically searched to identify patient-specific risk factors and clinical characteristics of EDKA in patients on SGLT-2i. The patient’s symptoms, clinical profile, laboratory results, and precipitants for EDKA were reviewed. Results: A total of 96 case reports identifying 116 patients with EDKA was fully reviewed. EDKA was twice prevalent in females (66.3%) than males (33.6%); median age was 52.15 ± 13.47, BMI was 29.3 ± 7.0. Among the 116 DKA events in SGLT-2i 92 (79.3%) were associated with Type-2 DM, 15 (12.9%) were Type-1 DM, 8 (6.9%) in LADA. Common symptoms were nausea (48.7%), vomiting (47%), and abdominal pain (28.2%). Canagliflozin was the most common SGLT-2i (40.5%), followed by Empagliflozin (29.3%) and Dapagliflozin (25.9%). The most common precipitant was surgery (17.2%), followed by infection (14.7%), fasting (11.2%), and Keto Diet (9.5%); others being reduced insulin use, alcoholism, and cancer. At presentation, average blood glucose was 196.8 ± 96.5, pH 7.1 ± 0.16, HCO3 8.7 ± 5.7 mmol/L, potassium 4.3 ± 1.03, anion-gap 24.2 ± 6.8 mmol/L, and the average HbA1C was 9.24 ± 2.08. Urine Ketones were positive in 81.89% of patients. 17 patients had pancreatic autoantibodies testing, and 7 were positive (41.2%) for glutamic acid decarboxylase-65 antibodies (anti-GAD-65). As a result, 7 patients were newly diagnosed with LADA who were previously misdiagnosed with type-2 DM. Conclusion: SGLT2i induced EDKA was found to be more predominant in females and type-2 DM. Diabetics should be educated on risk factors and consult physicians before commencing a dietary or exercise change. Physicians should be vigilant in diagnosing EDKA by thoughtful measurement of urine ketones and anti-GAD-65 testing can help diagnose underlying LADA.

Growth hormone directly favors hepatic ketogenesis in persons with prediabetes or type 2 diabetes mellitus treated with empagliflozin.

Sodium-glucose cotransporter 2 inhibitors increase glucagon secretion by pancreatic alpha cells and the susceptibility to ketoacidosis. On the other hand, growth hormone (GH) stimulates peripheral lipolysis and provides free fatty acids (FFA) for ketogenesis; however, it remains unresolved whether GH directly impacts hepatic ketogenesis. We aimed to investigate the role of physiologic GH levels in promoting ketogenesis in prediabetic or type 2 diabetic patients under empagliflozin treatment. Sixteen patients (11 women, 5 men) with prediabetes or type 2 diabetes mellitus, aged 55.6 ± 4.7 years and with a mean BMI of 30.7 ± 4.8 kg/m2 and HbA1c 7.1 ± 1.6% (means ± SD), participated in this study. All of them were submitted to three mixed-meal tests: they received placebo at -60 min (test 1), and empagliflozin 25 mg (test 2, 21st day) and empagliflozin 25 mg plus pegvisomant 30 mg were administered subcutaneously 36 h before (test 3, 28th day). After test 1, all patients were instructed to take empagliflozin 25 mg daily. The empagliflozin treatment decreased the plasma concentrations of glucose by 14% (P < 0.01), FFA by 23% (P < 0.01), and the insulin/glucagon ratio by 26% (P < 0.01), and it increased β-hydroxybutyrate by 44% (P < 0.05). The GH receptor block by pegvisomant restored the plasma β-hydroxybutyrate to baseline levels. We conclude that GH has a direct effect on promoting the ketogenesis environment in patients treated with empagliflozin.

Role of cAMP in Double Switch of Glucagon Secretion.

Glucose metabolism plays a crucial role in modulating glucagon secretion in pancreatic alpha cells. However, the downstream effects of glucose metabolism and the activated signaling pathways influencing glucagon granule exocytosis are still obscure. We developed a computational alpha cell model, implementing metabolic pathways of glucose and free fatty acids (FFA) catabolism and an intrinsically activated cAMP signaling pathway. According to the model predictions, increased catabolic activity is able to suppress the cAMP signaling pathway, reducing exocytosis in a Ca2+-dependent and Ca2+ independent manner. The effect is synergistic to the pathway involving ATP-dependent closure of KATP channels and consequent reduction of Ca2+. We analyze the contribution of each pathway to glucagon secretion and show that both play decisive roles, providing a kind of “secure double switch”. The cAMP-driven signaling switch plays a dominant role, while the ATP-driven metabolic switch is less favored. The ratio is approximately 60:40, according to the most recent experimental evidence.