Islet Α-cells Research Articles

Detection of Insulin in Insulin-Deficient Islets of Patients with Type 1 Diabetes.

Type 1 diabetes (T1D) is related to the autoimmune destruction of β-cells, leading to their almost complete absence in patients with longstanding T1D. However, endogenous insulin secretion persists in such patients as evidenced by the measurement of plasma C-peptide. Recently, a low level of insulin has been found in non-β islet cells of patients with longstanding T1D, indicating that other islet cell types may contribute to persistent insulin secretion. The present study aimed to test the ability of various antibodies to detect insulin in insulin-deficient islets of T1D patients. Pancreatic autopsies from two children with recent-onset T1D, two adults with longstanding T1D, and three control subjects were examined using double immunofluorescent labeling with antibodies to insulin, glucagon and somatostatin. Immunoreactivity to insulin in glucagon+ cells of insulin-deficient islets was revealed using polyclonal antibodies and monoclonal antibodies simultaneously recognizing insulin and proinsulin. Along with this, immunoreactivity to insulin was observed in the majority of glucagon+ cells of insulin-containing islets of control subjects and children with recent-onset T1D. These results suggest that islet α-cells may contain insulin and/or other insulin-like proteins (proinsulin, C-peptide). Future studies are needed to evaluate the role of α-cells in insulin secretion and diabetes pathogenesis.

TMEM55A-mediated PI5P signaling regulates α-cell actin depolymerization and glucagon secretion.

Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet α-cells, although the underlying mechanisms regulating glucagon secretion and α-cell dysfunction remain unclear. While insulin secretion from pancreatic β-cells has long been known to be partly controlled by intracellular phospholipid signaling, very little is known about the role of phospholipids in glucagon secretion. Here we show that TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-5-phosphate (PI5P), regulates α-cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse α-cells reduces exocytosis at low glucose, and this is rescued by the direct reintroduction of PI5P. This does not occur through an effect on Ca 2+ channel activity, but through a re-modelling of cortical F-actin dependent upon TMEM55A lipid phosphatase activity which occurs in response to oxidative stress. In summary, we reveal a novel pathway by which TMEM55A regulates α-cell exocytosis by manipulating intracellular PI5P level and the F-actin network.

Simultaneous High-speed Calcium Imaging Reveals Regulatory Paracrine Interactions of Islet α-cells With β- and δ-cells in Pancreas Tissue Slices

Simultaneous High-speed Calcium Imaging Reveals Regulatory Paracrine Interactions of Islet α-cells With β- and δ-cells in Pancreas Tissue Slices

The role of mu-opioid receptors in pancreatic islet α-cells.

Diabetes is a complex disease that impacts more than 500 million people across the world. Many of these individuals will develop diabetic neuropathy as a comorbidity, which is historically treated with exogenous opioids, such as morphine, oxycodone, or tramadol. Although these opioids are effective analgesics, growing evidence indicates that they may directly impact the endocrine pancreas function in patients. One common feature of these exogenous opioid ligands is their preference for the mu-opioid receptor (MOPR), so we aimed to determine whether endogenous MOPRs directly regulate pancreatic islet metabolism and hormone secretion. We show that pharmacological antagonism of MOPRs enhances glucagon secretion, but not insulin secretion, from human islets under high-glucose conditions. This increased secretion is accompanied by increased cAMP signaling. mRNA expression of MOPRs is robust in nondiabetic human islets but downregulated in islets from T2D donors, suggesting a link between metabolism and MOPR expression. Conditional genetic knockout of MOPRs in murine α-cells increases glucagon secretion under high-glucose conditions without increasing glucagon content. Consistent with downregulation of MOPRs during metabolic disease, conditional MOPR knockout mice treated with a high-fat diet show impaired glucose tolerance, increased glucagon secretion, increased insulin content, and increased islet size. Together, these results demonstrate a direct mechanism of action for endogenous opioid regulation of endocrine pancreas.

New Mechanisms to Prevent Heart Failure with Preserved Ejection Fraction Using Glucagon-like Peptide-1 Receptor Agonism (GLP-1 RA) in Metabolic Syndrome and in Type 2 Diabetes: A Review.

This review examines the impact of obesity on the pathophysiology of heart failure with preserved ejection fraction (HFpEF) and focuses on novel mechanisms for HFpEF prevention using a glucagon-like peptide-1 receptor agonism (GLP-1 RA). Obesity can lead to HFpEF through various mechanisms, including low-grade systemic inflammation, adipocyte dysfunction, accumulation of visceral adipose tissue, and increased pericardial/epicardial adipose tissue (contributing to an increase in myocardial fat content and interstitial fibrosis). Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is released from the enteroendocrine L-cells in the gut. GLP-1 reduces blood glucose levels by stimulating insulin synthesis, suppressing islet α-cell function, and promoting the proliferation and differentiation of β-cells. GLP-1 regulates gastric emptying and appetite, and GLP-1 RA is currently indicated for treating type 2 diabetes (T2D), obesity, and metabolic syndrome (MS). Recent evidence indicates that GLP-1 RA may play a significant role in preventing HFpEF in patients with obesity, MS, or obese T2D. This effect may be due to activating cardioprotective mechanisms (the endogenous counter-regulatory renin angiotensin system and the AMPK/mTOR pathway) and by inhibiting deleterious remodeling mechanisms (the PKA/RhoA/ROCK pathway, aldosterone levels, and microinflammation). However, there is still a need for further research to validate the impact of these mechanisms on humans.

Connections between body composition and dysregulation of islet α- and β-cells in type 2 diabetes

BackgroundAccompanying islet α- and β-cell dysregulation in type 2 diabetes (T2D) at the microscopic scale, alterations in body composition at the macroscopic scale may affect the pathogenesis of T2D. However, the connections between body composition and islet α-cell and β-cell functions in T2D have not been thoroughly explored.MethodsFor this cross-sectional study, we recruited a total of 729 Chinese Han patients with T2D in a consecutive manner. Dual-energy X-ray absorptiometry (DXA) was used to measure body composition, which included total bone-free mass, total fat and lean mass, trunk fat and lean mass and limb fat and lean mass. Every patient underwent an oral glucose tolerance test to simultaneously detect glucose, C-peptide and glucagon. The indices of islet α-cell function included fasting glucagon levels and the area under the curve of glucagon after a challenge (AUCglucagon), while the indices of β-cell function included the insulin sensitivity index derived from C-peptide (ISIC-peptide) and the area under the curve of C-peptide after a challenge (AUCC-peptide).ResultsAmong all patients, fat mass, especially trunk fat mass, was significantly correlated with ISIC-peptide and AUCC-peptide levels (r = − 0.330 and 0.317, respectively, p < 0.001), while lean mass, especially limb lean mass, was significantly correlated with fasting glucagon and AUCglucagon levels (r = − 0.196 and − 0.214, respectively, p < 0.001). Moreover, after adjusting for other relevant variables via multivariate linear regression analysis, increased trunk fat mass was independently associated with decreased ISIC-peptide (β = − 0.247, t = − 3.628, p < 0.001, partial R2 = 10.9%) and increased AUCC-peptide (β = 0.229, t = 3.581, p < 0.001, partial R2 = 8.2%), while decreased limb lean mass was independently associated with increased fasting glucagon (β = − 0.226, t = − 2.127, p = 0.034, partial R2 = 3.8%) and increased AUCglucagon (β = − 0.218, t = − 2.050, p = 0.041, partial R2 = 2.3%). Additionally, when separate analyses were performed with the same concept for both sexes, we found that increased trunk fat mass was still independently associated with decreased ISIC-peptide and increased AUCC-peptide, while decreased limb lean mass was still independently associated with increased fasting glucagon and AUCglucagon.ConclusionsIncreased trunk fat mass may partly account for decreased insulin sensitivity and increased insulin secretion, while decreased limb lean mass may be connected to increased fasting glucagon and postprandial glucagon secretion.

1768-P: Sel1L-Hrd1 Endoplasmic Reticulum-Associated Degradation Regulates PC2 Function and Proglucagon Processing in Islet α-Cells

Pancreatic islets rely on cellular protein quality control to maintain glucoregulatory hormone production. Proglucagon, precursor of glucagon and glucagon-like peptide 1 (GLP1), is expressed in islet α cells, intestinal L-cells, and some neurons. Islet α cells predominantly convert proglucagon to glucagon through prohormone convertase 2 (PC2). Islet-derived GLP1 is increasingly recognized as an important contributor to islet function, but the factors controlling its production remain unclear. To understand how protein quality control affects proglucagon processing in α cells, we inactivated the highly conserved Sel1L-Hrd1 endoplasmic reticulum-associated degradation (ERAD) system using Cre-loxP recombination in mice. Compared to littermate controls, Sel1LΔGcg mice showed an age-related decline in glucagon secretion in vivo, associated with a 43% decrease in pancreatic glucagon content (76.1 +/- 14.1 vs. 134.4 +/- 14.9 ng/g). Pancreatic GLP1 content was unchanged in Sel1LΔGcg mice (779.4 +/- 60.7 vs. 773.4 +/- 128.6 pg/g), despite a 37% reduction in α cell mass (0.19 +/- 0.04 vs. 0.12 +/- 0.04 mg/pancreas). Expression of the precursor form of PC2 (proPC2) was increased in Sel1LΔGcg α cells, suggesting accumulation of misfolded protein and/or impaired maturation. To investigate the mechanism underlying these changes, we used CRISPR to delete Sel1L in αTC cells. ERAD-deficient αTC-ΔSel1L cells showed 1.75-fold higher expression of proPC2, in addition to appearance of a novel truncated (~56 kD) form of proPC2. Though expression of the mature (64 kD) PC2 form was unchanged in αTC-ΔSel1L cells, PC2 activity was reduced by half in a specific enzyme assay. This was accompanied by decreased expression of 7B2, a chaperone required for proPC2 transit and activation in the secretory pathway. These data show that Sel1L-Hrd1 ERAD is required to ensure proper proPC2 maturation and glucagon production in α cells, thus maintaining classic α cell identity. Disclosure R.B.Reinert: None. W.Zhu: None. L.Pan: None. A.Russo: None. R.Ray: None. N.Shrestha: None. I.Lindberg: None. L.Qi: None. Funding National Institutes of Health (K08DK129719, T32DK007245)

1772-P: Alpha-Cell Heterogeneity Contributes to Phasic Glucagon Release

Glucagon, produced by α-cells of pancreatic islets, is one of the body’s primary tools for elevating blood glucose during hypoglycemia. The exact mechanism linking hypoglycemia to glucagon release by islet α-cells is, however, unclear; we therefore aimed to investigate it using optogenetics. We generated mice harboring the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in α-cells and imaged the effect of optically induced depolarization of plasma membrane (10-ms 470-nm light pulses at 20Hz) at the level of membrane potential, cytosolic Ca2+ ([Ca2+]i) and ATP/ADP ratio. At 1 mmol/L glucose, optostimulation depolarized the electrically active α-cells from -76mV to -8mV, induced a transient surge in [Ca2+]i and elevated glucagon secretion by 200%. In contrast, the glucagonostatic effect of high glucose (&amp;gt;6 mmol/L) was mitigated by optostimulation of α-cells. At 1 mmol/L glucose, the stimulatory effects were abolished by the KATP channel activator diazoxide (100μmol/L) and L-type Ca2+ channel blocker isradipine (10μmol/L). Notably, KATP channel inhibitor tolbutamide (100μmol/L) induced electrical activity in the fraction of α-cells insensitive to low glucose (35%). As stimulation of electrical activity persisted significantly beyond optoactivation, we probed the interplay between electrical activity and energy metabolism. The depolarizing stimulus induced by a cocktail of amino acids ((AA) 2 mmol/L of alanine + arginine +glutamine) or a moderate increase of extracellular K+ (from 4 to 15 mmol/L) reduced ATP/ADP in α-cells exhibiting spontaneous Ca2+ oscillations at low glucose. Surprisingly, in the population of inactive α-cells, the stimulation increased ATP/ADP ratio. These data indicate that α-cells are functionally heterogeneous due to metabolic differences. The observed secretory responses reflect the combination of stimulatory and inhibitory effects in electrically active and silent cells, which may contribute to the phasic nature of AA-induced glucagon secretion. Disclosure C.A.Miranda: None. H.Dou: None. J.Tolö: None. A.I.Tarasov: None. P.Rorsman: None. Funding Svenska Sällskapet för Medicinsk Forskning (PD20-0056); Swedish Vetenskap Rådet

Small molecule glucagon release inhibitors with activity in human islets.

Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet β-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 β-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Intestinal ACE2 regulates glucose metabolism in diet-induced obese mice through a novel gut-islet axis mediated by tryptophan.

Angiotensin-converting enzyme 2 (ACE2) plays a vital role in regulating intestinal tryptophan (Trp) transport and maintaining Trp homeostasis. This study aimed to investigate the functional relationship between intestinal ACE2 and Trp in the regulation of glucose metabolism under metabolic stress. ACE2-knockout mice and mice with adeno-associatedvirus-mediated overexpression of ACE2 were fed with a high-fat diet for 12 weeks to establish a high-fat-induced metabolic stress model. They were subjected to a Trp gavage intervention for another 4 weeks. Here, it is reported that ACE2 regulates intestinal Trp absorption by stabilizing neutral amino acid transporter B0 AT1. Notably, in ACE2-knockout mice, it was found that B0 AT1 and serum Trp levels were significantly reduced, which was not reversed by Trp supplementation. However, mice receiving adeno-associatedvirus-ACE2 did the opposite and showed significantly improved glycolipid metabolism. It was then confirmed that Trp potentiated glucagon-like peptide 1 production from intestinal and islet α-cells. Meanwhile, Trp-treated MIN6 cells ameliorated mitochondrial function and safely guarded MIN6 cells against reactive oxygen species exposure. This study highlights an essential role of ACE2 in the maintenance of systemic metabolism to optimize the function of the islets through a novel gut-islet axis mediated by Trp. These results provide proof-of-concept evidence for treating obesity and diabetes.

Studies on alpha-synuclein and islet amyloid polypeptide interaction.

Introduction: Parkinson's disease and type 2 diabetes have both elements of local amyloid depositions in their pathogenesis. In Parkinson's disease, alpha-synuclein (aSyn) forms insoluble Lewy bodies and Lewy neurites in brain neurons, and in type 2 diabetes, islet amyloid polypeptide (IAPP) comprises the amyloid in the islets of Langerhans. In this study, we assessed the interaction between aSyn and IAPP in human pancreatic tissues, both ex vivo and in vitro. Material and Methods: The antibody-based detection techniques, proximity ligation assay (PLA), and immuno-TEM were used for co-localization studies. Bifluorescence complementation (BiFC) was used for interaction studies between IAPP and aSyn in HEK 293 cells. The Thioflavin T assay was used for studies of cross-seeding between IAPP and aSyn. ASyn was downregulated with siRNA, and insulin secretion was monitored using TIRF microscopy. Results: We demonstrate intracellular co-localization of aSyn with IAPP, while aSyn is absent in the extracellular amyloid deposits. ASyn reactivity is present in the secretory granules of β-cells and some α-cells in human islets. The BiFC-expression of aSyn/aSyn and IAPP/IAPP in HEK293 cells resulted in 29.3% and 19.7% fluorescent cells, respectively, while aSyn/IAPP co-expression resulted in ∼10% fluorescent cells. Preformed aSyn fibrils seeded IAPP fibril formation in vitro, but adding preformed IAPP seeds to aSyn did not change aSyn fibrillation. In addition, mixing monomeric aSyn with monomeric IAPP did not affect IAPP fibril formation. Finally, the knockdown of endogenous aSyn did not affect β cell function or viability, nor did overexpression of aSyn affect β cell viability. Discussion: Despite the proximity of aSyn and IAPP in β-cells and the detected capacity of preformed aSyn fibrils to seed IAPP in vitro, it is still an open question if an interaction between the two molecules is of pathogenic significance for type 2 diabetes.

Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of β-cell identity and α-cell expansion in the mature pancreas

Abnormalities that characterize the pathophysiology of type 2 diabetes (T2D) include deficiencies of β-cells and the expansion of α-cells in pancreatic islets, manifested by lower insulin release and glucagon oversecretion. The molecular mechanisms that determine intra-islet interactions between pancreatic α- and β-cells are still not fully understood. The present study showed that stearoyl-coenzyme A (CoA) desaturase 1 (SCD1), an enzyme that is implicated in fatty acid metabolism, serves as a checkpoint in the control of endocrine cell equilibrium in pancreatic islets. Our data showed that SCD1 activity is essential for proper α-cell and β-cell lineage determination during morphogenesis of the pancreas and the maintenance of mature β-cell identity. The inhibition of SCD1 expression/activity led to both a decrease in the expression of β-cell signature genes (e.g., Pdx1, Nkx6.1, MafA, and Neurod1, among others) and induction of the expression of the dedifferentiation marker Sox9 in mature pancreatic islets. The transcriptional repression of Pdx1 and MafA in SCD1-deficient β-cells was related to the excessive methylation of promoter regions of these transcription factors. In contrast, SCD1 ablation favored the formation of α-cells over β-cells throughout pancreas organogenesis and did not compromise α-cell identity in adult pancreatic islets. Such molecular changes that were caused by SCD1 downregulation resulted in the mislocalization of α-cells within the core of islets and increased the ratio of pancreatic α- to β-cell mass. This was followed by islet dysfunction, including impairments in glucose-stimulated insulin release, simultaneously with elevations of basal glucagon secretion. Altogether, these findings provide additional mechanistic insights into the role of SCD1 in the pathogenesis of T2D.

Increased CD34 in pancreatic islet negatively predict islet β-cell decrease in type1 diabetes model.

Islet β-cell biomarkers can reflect changes in the number and function of islet β-cells in the prediabetes or early diabetes stage. CD34 is a commonly used stem cell biomarker; however, its expression and function in pancreatic islets remain unclear. In the present study, double immunofluorescence staining, proteomic bioinformatics analysis, and correlation analysis were used to explore the potential of CD34 as an islet β-cell biomarker. Bioinformatics analysis revealed that the amino acid sequence of CD34 was conserved among multiple species and abundantly expressed on mouse and human pancreatic tissues. Immunofluorescence demonstrated that in the control rat pancreas, CD34 was expressed on glucagon-labeled islet α-cells but not on insulin-labeled islet β-cells. Furthermore, the proportion of CD34-positive cells, which were also positive for glucagon, was significantly increased in alloxan-induced diabetes models. Statistical analysis revealed that the expression of CD34 was negatively correlated with the number of insulin-labeled islet β-cells during diabetes progression in dose-dependent fashion in alloxan-induced diabetes models. Furthermore, the results suggested that the transdifferentiation of islet β-cells into islet α-cells may occur in the process of diabetes. Thus, the present study demonstrated that CD34 is expressed on islet α-cells, and its number is linearly and negatively correlated with the number of islet β-cells, suggesting that CD34 can be used as a prospective biomarker for islet β-cells in the early diagnosis of diabetes. The study also suggests the transformation of β-cells to α-cells in diabetes which provide a potential to be applied towards diabetes mechanism research.

ODP296 An unusual presentation of Glucagonoma syndrome.

Abstract Introduction Glucagonoma is an uncommon neoplasm of the pancreatic neuroendocrine islet α-cells. At least 50% of cases will have metastatic disease when diagnosed. Glucagonomas can be associated with other tumors in Multiple Endocrine Neoplasia syndrome 1 (MEN 1), but this association is rare and comprises no more than 3% of glucagonomas. Glucagonoma syndrome is a rare paraneoplastic phenomenon characterized by necrolytic migratory erythema (NME), hyperglucagonemia, diabetes mellitus, anemia, weight loss, glossitis, cheilitis, steatorrhea, diarrhea, and venous thrombosis. Case presentation A 55-year-old Caucasian woman was admitted for acute onset of shortness of breath and she was seen by endocrinology team for persistent hyperglycemia. Her medical history revealed a long-standing uncontrolled type 2 diabetes mellitus and an episode of left lower extremity deep-vein thrombosis. Her physical exam revealed tachycardia, otherwise unremarkable, with no skin lesions. Laboratory results showed hyperglycemia (387 mg/dl), Hemoglobin A1C, 9%, and hypoalbuminemia (3.4 g/dl). Her Hemoglobin (13 g/dl), white cell count (6100/μL), and platelet level (19.3 × 104/μL) were within normal limits. The patient underwent CT pulmonary angiogram, which showed a large saddle pulmonary embolism as well as a distal pancreatic mass measuring 4.5×3.4 cm, which contains numerous coarse calcifications. Pancreatic mass was confirmed in a CT abdomen and pelvis with contrast. CA 19-9 was elevated to 51 U/ml (normal range, 0- 35U/ml). The GI team performed an Endoscopic Ultrasound, and samples were sent for FNA and flow cytometry which revealed a 4 cm irregular heterogeneous mass with calcifications at the distal pancreatic body, positive for Pankeratin (CK), Cam 5.2, Synaptophysin, Chromogranin A, and CD56, consistent with Pancreatic neuroendocrine Tumor. Her serum glucagon level was elevated to 447 pg/mL (normal range, 50 -150 pg/mL), while her levels of other hormones, such as somatostatin or gastrin, were within normal limits. Glucagonoma of the pancreas was diagnosed, and a spleen-preserving distal pancreatectomy was performed. Histopathological examination revealed a 5.9 cm alpha-cell pancreatic tumor without lymphovascular or perineural tumor invasion. (AJCC 8th edition staging II, pT3, pN0, MX, G1). Immunohistochemical staining was strongly positive for glucagon. A gallium-68 positron emission tomography (68Ga-PET, Netspot) did not show metastasis. Post resection surveillance showed normalization in Glucagon level and no evidence of recurrence in CT abdomen. Conclusion The diagnosis of glucagonoma is often delayed due to unusual initial manifestations of glucagonoma. Early diagnosis provides a good chance of complete surgical removal as the only curative treatment. Presentation: No date and time listed

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Metformin and Gegen Qinlian Decoction boost islet α-cell proliferation of the STZ induced diabetic rats

BackgroundThe traditional Chinese medicine Gegen Qinlian Decoction (GQD), as well as metformin, had been reported with anti-diabetic effects in clinical practice.ObjectiveTo verify whether these two medicines effectively ameliorate hyperglycemia caused by deficiency of islet β-cell mass which occurs in both type 1 and type 2 diabetes.MethodsSD rats were injected with a single dose of STZ (55 mg/kg) to induce β-cell destruction. The rats were then divided into control, diabetes, GQD and metformin group. GQD and metformin groups were administered with GQD extract or metformin for 6 weeks. The islet α-cell or β-cell mass changes were tested by immunohistochemical and immunofluorescent staining. The potential targets and mechanisms of GQD and metformin on cell proliferation were tested using in silico network pharmacology. Real-time PCR was performed to test the expression of islet cells related genes and targets related genes.ResultsBoth GQD and metformin did not significantly reduce the FBG level caused by β-cell mass reduction, but alleviated liver and pancreas histopathology. Both GQD and metformin did not change the insulin positive cell mass but increased α-cell proliferation of the diabetic rats. Gene expression analysis showed that GQD and metformin significantly increased the targets gene cyclin-dependent kinase 4 (Cdk4) and insulin receptor substrate (Irs1) level.ConclusionThis research indicates that GQD and metformin significantly increased the α-cell proliferation of β-cell deficiency induced diabetic rats by restoring Cdk4 and Irs1 gene expression.

1448-P: Partial Pancreatectomy Increased α-Cell Volume Density, but Not Compensated ß-Cell Volume in Japanese Subjects

In type 2 diabetes, deficit of insulin secretion is often ascribed to the reduction of β-cell volume, implying that regeneration of β-cells could be one of its radical treatments. Hitherto, several models are known to elicit β-cell compensation in rodents. Among them, application of partial pancreatectomy can induce replenishment of β-cell volume in young mice, while it is unclear whether this occurs in humans. In the present study, islet pathology was evaluated using the pancreatic specimens from the patients with pancreatic cancer who underwent repeated (i.e., partial followed by total) pancreatectomy because of its local recurrence after the initial surgery. Ten subjects (5 males and 5 females) at Hirosaki University Hospital or Chiba University Hospital were evaluated. Morphometric analysis was performed on formalin fixed pancreatic sections, in which the quadruple immunostaining for each hormone and the fluorescent immunostaining for ALDH1A3 was conducted. The mean age at the first and second operations was 66.2±and 71.6±5.8 years old, respectively. The mean duration until recurrence was 3.2±2.0 years. There were no significant differences in HbA1c and BMI between first and second preoperation. In morphometrical analysis, α-cell volume in the second surgery was significantly increased compared to the first surgery (p&amp;lt;0.04) , while β-cell volume was comparable. There was a positive correlation between the frequency of conduit glucagon positive cells and islet α-cell volume (r=0.66, p&amp;lt;0.05) . The frequency of ALDH1A3 positive cells were positively correlated with the α-cell/ β-cell ratio in the second surgical specimen (r=0.81, p&amp;lt;0.01) . Taken together, unlike rodents, our results indicate that partial pancreatectomy did not induce replenishment of β-cell volume, but increased α-cell volume in human subjects. As its mechanism, the neogenesis of ductal endocrine cells and/or the transdifferentiation of β-cells to α-cells in the islets may be involved. Disclosure Y.Takeuchi: None. H.Mizukami: None. S.Osonoi: None. K.Kudoh: None. T.Sasaki: None. S.Yagihashi: None.

GABA and insulin but not nicotinamide augment α- to β-cell transdifferentiation in insulin-deficient diabetic mice

AimPoorly controlled diabetes is characterised by a partial or complete loss of pancreatic islet β-cells, which deprives the remaining islet cells of important β-cell-derived soluble signals, such as insulin or GABA. We aimed to dissect the role of the two signals in the development of islet α-cells, focusing specifically on α-/β-cell transdifferentiation and using the stem cell differentiation factor nicotinamide as a comparator. MethodsStreptozotocin (STZ)-treated diabetic mice expressing a fluorescent reporter in pancreatic islet α-cells were injected with GABA (10 mg/kg once daily), nicotinamide (150 mg/kg once daily) or insulin (1U/kg three times daily) for 10 days. The impact of the treatment on metabolic status of the animals as well as the morphology, proliferative potential and transdifferentiation of pancreatic islet cells was assessed using biochemical methods and immunofluorescence. ResultsMetabolic status of STZ-diabetic mice was not dramatically altered by the treatment interventions, although GABA therapy did reduce circulating glucagon and augment pancreatic insulin stores. The effects of the exogenous agents on islet β-cells ranged from the attenuation of apoptosis (insulin, nicotinamide) to enhancement of proliferation (GABA). Furthermore, insulin and GABA but not nicotinamide enhanced the differentiation of α-cells into β-cells and increased relative number of ‘bihormonal’ cells, expressing both insulin and glucagon. ConclusionsOur data suggest a role for endogenous insulin and GABA signalling in α-cell plasticity, which is likely to bypass the common nicotinamide-sensitive stem cell differentiation pathway.

The evolving story of incretins (GIP and GLP-1) in metabolic and cardiovascular disease: A pathophysiological update.

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have their main physiological role in augmenting insulin secretion after their nutrient-induced secretion from the gut. A functioning entero-insular (gut-endocrine pancreas) axis is essential for the maintenance of a normal glucose tolerance. This is exemplified by the incretin effect (greater insulin secretory response to oral as compared to "isoglycaemic" intravenous glucose administration due to the secretion and action of incretin hormones). GIP and GLP-1 have additive effects on insulin secretion. Local production of GIP and/or GLP-1 in islet α-cells (instead of enteroendocrine K and L cells) has been observed, and its significance is still unclear. GLP-1 suppresses, and GIP increases glucagon secretion, both in a glucose-dependent manner. GIP plays a greater physiological role as an incretin. In type 2-diabetic patients, the incretin effect is reduced despite more or less normal secretion of GIP and GLP-1. While insulinotropic effects of GLP-1 are only slightly impaired in type 2 diabetes, GIP has lost much of its acute insulinotropic activity in type 2 diabetes, for largely unknown reasons. Besides their role in glucose homoeostasis, the incretin hormones GIP and GLP-1 have additional biological functions: GLP-1 at pharmacological concentrations reduces appetite, food intake, and-in the long run-body weight, and a similar role is evolving for GIP, at least in animal studies. Human studies, however, do not confirm these findings. GIP, but not GLP-1 increases triglyceride storage in white adipose tissue not only through stimulating insulin secretion, but also by interacting with regional blood vessels and GIP receptors. GIP, and to a lesser degree GLP-1, play a role in bone remodelling. GLP-1, but not GIP slows gastric emptying, which reduces post-meal glycaemic increments. For both GIP and GLP-1, beneficial effects on cardiovascular complications and neurodegenerative central nervous system (CNS) disorders have been observed, pointing to therapeutic potential over and above improving diabetes complications. The recent finding that GIP/GLP-1 receptor co-agonists like tirzepatide have superior efficacy compared to selective GLP-1 receptor agonists with respect to glycaemic control as well as body weight has renewed interest in GIP, which previously was thought to be without any therapeutic potential. One focus of this research is into the long-term interaction of GIP and GLP-1 receptor signalling. A GLP-1 receptor antagonist (exendin [9-39]) and, more recently, a GIP receptor agonist (GIP [3-30] NH2 ) and, hopefully, longer-acting GIP receptor agonists for human use will be helpful tools to shed light on the open questions. A detailed knowledge of incretin physiology and pathophysiology will be a prerequisite for designing more effective incretin-based diabetes drugs.

Mesenchymal stem cell-conditioned medium alleviates high fat-induced hyperglucagonemia via miR-181a-5p and its target PTEN/AKT signaling

Backgroundα-cell dysregulation gives rise to fasting and postprandial hyperglycemia in type 2 diabetes mellitus(T2DM). Administration of Mesenchymal stem cells (MSCs) or their conditioned medium can improve islet function and enhance insulin secretion. However, studies showing the direct effect of MSCs on islet α-cell dysfunction are limited. MethodsIn this study, we used high-fat diet (HFD)-induced mice and α-cell line exposure to palmitate (PA) to determine the effects of bone marrow-derived MSC-conditioned medium (bmMSC-CM) on glucagon secretion. Plasma and supernatant glucagon were detected by enzyme-linked immunosorbent assay(ELISA). To investigate the potential signaling pathways, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), AKT and phosphorylated AKT(p-AKT) were assessed by Western blotting. ResultsIn vivo, bmMSC-CM infusion improved the glucose and insulin tolerance and protected against HFD-induced hyperglycemia and hyperglucagonemia. Meanwhile, bmMSC-CM infusion ameliorated HFD-induced islet hypertrophy and decreased α- and β-cell area. Consistently, in vitro, glucagon secretion from α-cells or primary islets was inhibited by bmMSC-CM, accompanied by reduction of intracellular PTEN expression and restoration of AKT signaling. Previous studies and the TargetScan database indicate that miR-181a and its target PTEN play vital roles in ameliorating α-cell dysfunction. We observed that miR-181a-5p was highly expressed in BM-MSCs but prominently lower in αTC1-6 cells. Overexpression or downregulation of miR-181a-5p respectively alleviated or aggravated glucagon secretion in αTC1-6 cells via the PTEN/AKT signaling pathway. ConclusionsOur observations suggest that MSC-derived miR-181a-5p mitigates glucagon secretion of α-cells by regulating PTEN/AKT signaling, which provides novel evidence demonstrating the potential for MSCs in treating T2DM.