Normal Islets Research Articles

Unveiling islet heterogeneity using an automated microfluidic imaging system

Islets of Langerhans are a therapeutic target for diabetes and prediabetes. Measurements of therapeutic inhibitory or excitatory concentrations are often performed using large groups of islets, however, the population heterogeneity cannot be observed when examining the ensemble response. Normal islet function and islet response to therapeutic treatment can be affected by islet heterogeneity, influencing the progression of diabetes mellitus. To identify heterogeneity in an islet population, we developed a simple microfluidic device capable of delivering a stimulant to four independent chambers, allowing measurements of individual responses from a population of 20–25 islets. The device enabled accurate delivery of the same stimulant concentration to all four chambers, with an error <1% between chambers. To demonstrate the capability of this system, ensemble and individual EC/IC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} measurements of glucose and diazoxide were performed on murine islets. Results showed little heterogeneity of glucose EC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} values with all 21 islets within ± 0.6 mM of the ensemble value of 7.4 mM. In contrast, application of diazoxide to 24 islets in the presence of 20 mM glucose resulted in 37% of islets having an IC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} greater than 10% from the ensemble value of 10.2 μM. The simple system developed here is amenable to further studies on islet heterogeneity, and is applicable to investigate heterogeneity in other cell types.

Characterization of Human Pancreatic Islet Cells using a Single-Cell Western Blot Platform.

Islet transplantation is an effective treatment for type 1 diabetes. However, transplant success depends on quick islet assessment because islets deteriorate 2-3 days after isolation. A new tool, single-cell Western blot (scWestern), offers results within one day. In this study, we aimed to test the suitability of scWestern to detect protein markers for beta (insulin), alpha (glucagon), and delta (somatostatin) cells, the three major endocrine cell types in islets. We characterized the antibody specificity, signal intensity, and cell identification on the scWestern platform, and then compared the islet cell composition analysis between scWestern and immunohistochemistry performed by the Integrated Islet Distribution Program (IIDP). Islet cell composition is comparable for alpha and beta cells, but not delta cells. Protein expression levels of insulin, glucagon, and somatostatin in individual islet cells varied greatly, highlighting cell type heterogeneity. Surprisingly, scWestern revealed double-hormonal cells (~1%), co-expressing insulin and somatostatin or insulin and glucagon, in non-diabetic and non-obese adult human islets, which was confirmed by confocal immunofluorescence microscopy. These results demonstrate that each alpha, beta, and delta cells express varying levels of peptide hormones, and a small subpopulation co-expresses double hormones in normal human islets. The scWestern platform will enable timely assessment of beta cell mass in isolated islets before clinical transplantation.

Single-cell Transcriptomics, Web-based Systems Pharmacology and Integrated Transcriptomics Network Analysis Identified Diagnostic Targets and Drug Candidates for Type 2 Diabetes.

To discover new therapeutic targets for Type 2 diabetes (T2D) and develop a new diagnostic model. T2D is a chronic disease that can be controlled by oral hypoglycemic drugs, however, it cannot be fully cured. The continued increase in the prevalence of T2D and the limitations of existing treatments urgently call for the development of new drugs to be able to effectively control the progression of the disease. We aimed to discover new therapeutic targets for T2D and to develop a new diagnostic model. Single-cell transcriptome, web-based systematic pharmacology, and transcriptology were applied to identify T2D diagnostic targets and drug candidates and to analyze the underlying molecular mechanisms. By single-cell clustering analysis, we identified seven subsets between the normal islet β-cell samples and T2D islet β-cell samples. A total of 27 key genes in the intersection of insulin- related genes and diabetes-related genes were selected by protein-protein interaction (PPI) analysis and MolecularComplexDetection (MCODE) analysis. Notably, ESR1, MME, and CCR5 had the area under curves (AUC) values as high as 67.95%, 66.67%, and 66.03% for the diagnosis of T2D, respectively. Since the expression of MME in T2D samples was significantly higher than in normal samples, we screened 155 drug candidates against MME in T2D. Finally, the molecular docking revealed a strong binding strength between MME and DB05490, which was one of the most effective candidate drugs for treating T2D. Our study screens for diagnostic signatures and potential therapeutic agents for T2D, which provides valuable insights into the development of T2D biomarkers and their drug discovery.

PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

Ginseng extract improves pancreatic islet injury and promotes β-cell regeneration in T2DM mice.

Panax ginseng C. A. Mey. (Araliaceae; Ginseng Radix et Rhizoma), a traditional plant commonly utilized in Eastern Asia, has demonstrated efficacy in treating neuro-damaging diseases and diabetes mellitus. However, its precise roles and mechanism in alleviating type 2 diabetes mellitus (T2DM) need further study. The objective of this study is to explore the pharmacological effects of ginseng extract and elucidate its potential mechanisms in protecting islets and promoting β-cell regeneration. The T2DM mouse model was induced through streptozotocin combined with a high-fat diet. Two batches of mice were sacrificed on the 7th and 28th days following ginseng extract administration. Body weight, fasting blood glucose levels, and glucose tolerance were detected. Morphological changes in the pancreatic islets were examined via H & E staining. Levels of serum insulin, glucagon, GLP-1, and inflammatory factors were measured using ELISA. The ability of ginseng extract to promote pancreatic islet β-cell regeneration was evaluated through insulin & PCNA double immunofluorescence staining. Furthermore, the mechanism behind β-cells regeneration was explored through insulin & glucagon double immunofluorescence staining, accompanied by immunohistochemical staining and western blot analyses. The present research revealed that ginseng extract alleviates symptoms of T2DM in mice, including decreased blood glucose levels and improved glucose tolerance. Serum levels of insulin, GLP-1, and IL-10 increased following the administration of ginseng extract, while levels of glucagon, TNF-α, and IL-1β decreased. Ginseng extract preserved normal islet morphology, increased nascent β-cell population, and inhibited inflammatory infiltration within the islets, moreover, it decreased α-cell proportion while increasing β-cell proportion. Mechanistically, ginseng extract might inhibit ARX and MAFB expressions, increase MAFA level to aid in α-cell to β-cell transformation, and activate AKT-FOXM1/cyclin D2 to enhance β-cell proliferation. Our study suggests that ginseng extract may be a promising therapy in treating T2DM, especially in those with islet injury.

Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Induction of islet autoimmunity to defective ribosomal product of the insulin gene as neoantigen after anti-cancer immunotherapy leading to autoimmune diabetes.

The autoimmune response in type 1 diabetes (T1D), in which the beta cells expressing aberrant or modified proteins are killed, resembles an effective antitumor response. Defective ribosomal protein products in tumors are targets of the anti-tumor immune response that is unleashed by immune checkpoint inhibitor (ICI) treatment in cancer patients. We recently described a defective ribosomal product of the insulin gene (INS-DRiP) that is expressed in stressed beta cells and targeted by diabetogenic T cells. T1D patient-derived INS-DRiP specific T cells can kill beta cells and are present in the insulitic lesion. T cells reactive to INS-DRiP epitopes are part of the normal T cell repertoire and are believed to be kept in check by immune regulation without causing autoimmunity. T cell autoreactivity was tested using a combinatorial HLA multimer technology measuring a range of epitopes of islet autoantigens and neoantigen INS-DRiP. INS-DRiP expression in human pancreas and insulinoma sections was tested by immunohistochemistry. Here we report the induction of islet autoimmunity to INS-DRiP and diabetes after ICI treatment and successful tumor remission. Following ICI treatment, T cells of the cancer patient were primed against INS-DRiP among other diabetogenic antigens, while there was no sign of autoimmunity to this neoantigen before ICI treatment. Next, we demonstrated the expression of INS-DRiP as neoantigen in both pancreatic islets and insulinoma by staining with a monoclonal antibody to INS-DRiP. These results bridge cancer and T1D as two sides of the same coin and point to neoantigen expression in normal islets and insulinoma that may serve as target of both islet autoimmunity and tumor-related autoimmunity.

Structural modifications of INGAP-PP present in HTD4010 peptide potentiate its effect on rat islet gene expression and insulin secretion.

Type 2 diabetes (T2D) is characterized by peripheral insulin resistance and altered insulin secretion due to a progressive loss of β-cell mass and function. Today, most antidiabetic agents are designed to resolve impaired insulin secretion and/or insulin resistance, and only GLP-1-based formulations contribute to stopping the decline in β-cell mass. HTD4010, a peptide carrying two modifications of the amino acid sequence of INGAP-PP (N-terminus acetylation and substitution of Asn13 by Ala) showed greater plasma stability and could be a good candidate for proposal as a drug that could improve β cell mass and function lost in T2D. In the present study, we showed that HTD4010 included in the culture media of normal rat islets at a dose 100 times lower than that used for INGAP-PP was able to modulate, in the same way as the original peptide, both insulin secretion in response to glucose and the expression of key genes related to insular function, insulin and leptin intracellular pathways, neogenesis, apoptosis, and inflammatory response. Our results confirm the positive effect of HTD4010 on β-cell function and gene expression of factors involved in the maintenance of β-cell mass. Although new assays in animal models of prediabetes and T2D must be performed to be conclusive, our results are very encouraging, and they suggest that the use of HTD4010 at a dose 100 times lower than that of INGAP-PP could minimize its side effects in a future clinical trial.

Systemic lupus erythematosus and diabetic mellitus is it single or two entity(es)

Introduction: Systemic lupus erythematosus (SLE) is a complex and chronic autoimmune disease with varied clinical manifestations and multi-organ involvement. The disease may present with acute, severe, and life-threatening symptoms or present as a fluctuating and chronic process that impacts several systems and increases the risk of malignancy. Systemic lupus erythematosus (SLE) often coexists with other diseases. Diabetes mellitus (DM) is an example and patients with SLE-DM can present with clinical features common to both disorders. Type 2 diabetes (T2D) is characterized by insulin resistance the inability of tissues to respond to insulin, and a progressive pancreatic beta cell dysfunction in response to glucose levels. The disease is thought to be caused by environmental and inherited factors in about equal proportions. Many environmental risk factors are known, including obesity, sedentary lifestyle, stress, nutritional factors and toxins. The report aims to describe Systemic Lupus Erythematosus and Diabetes mellitus as single or two entity(es). Case presentation: A 14-year-old female patient who was admitted to the hospital with complaints of weakness and drowsiness was getting worse 3 days before admitted from hospital. There have been complaints of fatigue for last 3 years accompanied by a lot of eating and drinking and urinating. Complaints of pain in both joints of the hands and both knees appeared in the last 3 months and had been getting worse. Hair loss existed in the past 4 months before being admitted to the hospital. Blurred vision in the right eye in the last 6 months has worsened in the last 1 month. Vision loss in the last 3 months. Body weight was reduced 4 kilograms within 3 months without any loss of appetite. Initial laboratory results revealed hyperglycemia, antinuclear antibodies (ANA) IF 1:100 and ANA Profile Sm (+), dyslipidemia, increase of creatinine serum with glomerular filtration rate 72.41 m2/BSA (decreased 38%). Other examinations showed that C-peptide is normal and beta islet antibody is negative. Echocardiography resulted tricuspid regurgitation with normal ventricle function. Conclusion: Systemic Lupus Erythematosus and Diabetes mellitus is a rare condition that warrants a thorough evaluation to both determine the diagnosis and predict the clinical course of disease.

GAD2 is a highly specific marker for neuroendocrine neoplasms of the pancreas

Abstract Introduction/Objective Glutamate decarboxylase 2 (GAD2) is the most important inhibitory neurotransmitter and plays a role in insulin-producing β-cells of pancreatic islets. The limitation of GAD2 expression to normal brain and pancreatic islet cells makes GAD2 a potential immunohistochemical diagnostic marker. Methods/Case Report To evaluate the diagnostic utility of GAD2 immunohistochemistry (IHC), a tissue microarray containing 19,202 samples from 152 different tumor entities and 608 samples of 76 different normal tissue types was analyzed. Data on progesterone receptor (PR) expression were available on 15,232 tumors from a previous study. Results (if a Case Study enter NA) GAD2 positivity occurred in 20 of 152 tumor categories including 5 tumor categories with at least one strongly positive case. GAD2 positivity was most frequent in neuroendocrine carcinomas (58.3%) and neuroendocrine tumors (63.2%) of the pancreas, followed by granular cell tumors (37.0%) and neuroendocrine tumors of the lung (11.1%). GAD2 in &amp;lt;10% of cases occurred in 16 other tumor entities including paraganglioma, medullary thyroid carcinoma, and small cell carcinoma of the urinary bladder. In a cohort of 95 pancreatic and 380 extra-pancreatic neuroendocrine neoplasms, GAD2 had a sensitivity of 64.2% and a specificity 96.3% for pancreatic tumor origin while PR had a sensitivity of 56.8% and a specificity of 92.6%. Conclusion GAD2 IHC is a highly useful diagnostic tool for the identification of pancreatic origin in case of neuroendocrine neoplasms with unknown site of origin. The particular strength of GAD2 is its high specificity for pancreatic origin. For the distinction of a pancreatic tumor origin, both sensitivity and specificity is higher for GAD2 than for PR. The combination of PR and GAD2 further increases sensitivity and specificity.

NADH intraperitoneal injection prevents massive pancreatic beta cell destruction in a streptozotocin-induced diabetes in rats.

Diabetes mellitus is a chronic metabolic disease characterized by persistent hyperglycemia, revealing a decrease in insulin efficiency. The sustained glucotoxic pancreatic microenvironment increases reactive oxygen species generation, resulting in chronic oxidative stress responsible for massive DNA damage. This triggers PARP-1 activation with both NAD+ and ATP depletion, affecting drastically pancreatic beta cells' energy storage and leading to their dysfunction and death. The aim of the present study is to highlight the main histological changes observed in pancreatic islets pre-treated with a unique NADH intraperitoneal injection in a streptozotocin-(STZ)-induced diabetes model. In order to adjust NADH doses, a preliminary study with three different doses, 500mg/kg, 300mg/kg, and 150mg/kg, respectively, was conducted. Subsequently, and on the basis of the results of the aforementioned study, Wistar rats were randomly divided into four groups: non-diabetic control group, diabetics (STZ 45mg/kg), NADH-treated group (150mg/kg) 15min before STZ administration, and NADH-treated group (150mg/kg) 15min after STZ administration. The effect of NADH was assessed by blood glucose level, TUNEL staining, histo-morphological analysis, and immunohistochemistry. The optimum protective dose of NADH was 150mg/kg. NADH effectively decreased hyperglycemia and reduced diabetes induced by STZ. Histologically, NADH pre-treatment revealed a decrease in beta cell death favoring apoptosis over necrosis and therefore preventing inflammation with further beta cell destruction. Our data clearly demonstrate that NADH prior or post-treatment could effectively prevent the deleterious loss of beta cell mass in STZ-induced diabetes in rats and preserve the normal pancreatic islet's function.

OR16-03 Epigenetic Studies Of Pancreatic Neuroendocrine Tumours (PNETs) Reveal Decreased DNA Hydroxymethylation In Non-functioning PNETs But Not Insulinomas

Abstract Disclosure: K.A. English: None. A. Selberherr: None. O.A. Shariq: None. E. O'Neill: None. K.E. Lines: None. R.V. Thakker: None. Background: DNA methylation and hydroxymethylation (5hmC) are epigenetic alterations that can silence or maintain gene transcription, respectively. Pancreatic neuroendocrine tumours (PNETs), which can be hormone-secreting (e.g. insulinomas) or non-functioning (NF-PNETs), have a low mutational burden and are reported to be associated with DNA hypermethylation, but the role of 5hmC in PNETs is unknown. Aim: To determine the patterns of DNA 5hmC in NF-PNETs, insulinomas, and normal pancreatic islets. Methods: NF-PNETs (n=31, mean age 54 ± 13 years (y), 14 males (M), tumour grade (G) G1=15, G2=13, G3=3) and insulinomas (n=21, mean age 50 ± 21y, 9M, G1=17 and G2=4) were collected from patients who did not have a genetic syndrome associated with PNETs together with 14 normal pancreata (mean age 63 ± 12y, 11M). Samples were obtained following ethical approval from the Universities of Oxford and Vienna. DNA 5hmC was assessed by immunohistochemistry (IHC) and MethylationEPIC array (that interrogates 5hmC CpG sites). Multivariate analysis was used to determine variables associated with 5hmC. Transcriptome analysis was undertaken by RNA-Seq. Results: NF-PNETs had reduced 5hmC when compared to insulinomas (p&amp;lt;0.01) and to normal adjacent islets (p&amp;lt;0.001). Thus, 5hmC was ∼3 fold lower in NF-PNETs compared to normal adjacent islets (n=12; p&amp;lt;0.001), whilst 5hmC between insulinomas and normal adjacent islets (n=15) was not significantly different. Multivariate analysis confirmed this finding, with NF-PNETs being associated with lower 5hmC when controlled for patient age, sex and tumour grade (p&amp;lt;0.01). In normal pancreatic tissue 5hmC was ∼2 fold higher in normal islets when compared to exocrine tissue (51 islets assessed in total, range 1-9 islets/sample from 34 fields of 305,866um2 studied; p&amp;lt;0.001). MethylationEPIC array analysis, using DNA from NF-PNETs (n=5), insulinomas (n=4) and normal pancreatic tissue (n=7), revealed NF-PNETs and insulinomas to have a significant decrease in 5hmC compared to normal pancreatic tissue (p&amp;lt;0.001). Furthermore, subtype analysis revealed that NF-PNETs had lower 5hmC compared to insulinomas (0% vs 0.003%, adjusted p&amp;lt;0.08). To determine possible mechanisms underlying these differences in tumour subtype 5hmC, RNA-Seq analysis using NF-PNETs (n=7) and insulinomas (n=6) was performed. This revealed 79 genes to be differentially expressed (adjusted p&amp;lt;0.01), of which 33 were upregulated (including Calcium/Calmodulin Dependent Protein Kinase 2 Gamma (CAMK2G)); and 46 were down-regulated (including Glucagon Like peptide 1 receptor (GLP1R)) in NF-PNETs. Interestingly, CAMK2G and GLP1R are both involved in DNA 5hmC modulation via cAMP. Conclusions: DNA 5hmC, an epigenetic mechanism, is significantly lower in NF-PNETs when compared to normal islets and insulinomas and may involve dysregulation of cAMP-mediated signalling pathways. Presentation: Friday, June 16, 2023

SAT064 Establishment And Initial Characterization Of Two Parallel Targeted Human ZnT8 Knock-in Murine Lines For Studying The Type 2 Diabetes Risk Variant At SNP rs13266634 Of The ZnT8 Gene

Abstract Disclosure: J.C. Wang: None. C. Adelma: None. M.G. Hayes: None. M. El Muayed: None. Background: Studies showed an association between R variant at position 325 in the β-cell zinc transporter ZnT8 and an increased risk for developing type 2 diabetes mellitus (T2DM). However, the mechanism by which this variant confers an increased risk for type 2 diabetes remains unclear. Methods: We generated a murine model for studying the effect of SNP rs13266634 on β-cell physiology. This model consists of two parallel murine lines in which conditional (RIP-Cre induced) expression constructs for the human variants of SLC30A8 (ZnT8), namely R (hZnT8R325, T2DM risk allele) and W (hZnT8W325, non-risk allele) of SNPrs13266634 were inserted into the rosa26 locus via targeted integration. β-cell specific expression of each of ZnT8R325 and ZnT8W325 was induced by cross breeding with a RIP-Cre line, expressing Cre recombinase in β-cells. All mouse lines were backcrossed into a C57BL/6N background null for the mouse variant of ZnT8. Islet zinc concentrations was measured by ICP-MS following hydrolysis in HNO3. Ex-vivo Glucose Stimulated Insulin Secretion Ratio (GSISR) was measured by incubation of isolated islets in 2.8 mM glucose, followed by 16.7 mM glucose minutes after an initial 60 min equilibration phase and calculating the insulin secretion at 16.7 mM/2.8 mM glucose. Data are reported as averages ± SEM from 3 independent experiments (n=3), each with 4 technical replicates. Results: Expression of both variants was verified by mRNA PCR from islets. Levels of expression in both lines were identical, owing to the targeted integration approach. Expression of each of hZnT8R325 and ZnT8W325 resulted in restoration of islet zinc concentration to levels similar to those found in normal human islets and wild type mouse islets. This confirms proper function of the hZnT8R325 transgenes in this newly established model. hZnT8R expressing islets showed a trend to lower insulin response to ex-vivo glucose stimulation compared to hZnT8W325 expressing islets (GSISR- 1.37 ± 0.41 vs 1.97 ± 0.32 respectively), as well as Cre expressing (GSISR 1.53 ± 0.40) or inactive construct controls (GSISR 2.43 ± 0.57). In-vivo glucose stimulated insulin secretion was not significantly different between hZnT8R325 expressing mice and those expressing hZnT8W325. As previously reported by others, all mice with β-cell Cre expression (RIP-Cre activated hZnT8 expressing lines and RIP-Cre controls) showed a lower insulin secretory response to glucose compared to non-Cre expressing mice (inactive construct controls). Conclusion: The newly created murine mouse model expresses functional human homologues of the ZnT8 variants hZnT8R325 and hZnT8W325 that result in restored beta cell zinc uptake. A trend to lower insulin secretory capacity was observed in ex-vivo glucose stimulated insulin secretion experiments. This model will be valuable tool to elucidate the functional consequences of the T2DM risk variant at SNP rs13266634. Presentation: Saturday, June 17, 2023

Pancreatic Cancer-Derived Exosomal miR-Let-7b-5p Stimulates Insulin Resistance in Skeletal Muscle Cells Through RNF20/STAT3/FOXO1 Axis Regulation.

Cancers trigger systemic metabolic disorders usually associated with glucose intolerance, which is an initially apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). Our previous study reported that mice pancreatic cancer-derived exosomes could induce skeletal muscle cells (C2C12) IR, and exosomal microRNAs (miRNAs) may exert an important effect. However, the underlying mechanism remains to be further elucidated. qPCR was used to determine the expression of let-7b-5p in normal pancreatic islet cells and PC cells. Exosomes were purified from PC cell culture medium by ultracentrifugation. The role let-7b-5p on IR-mediated by PC cells-derived exosomes was asses by Oil Red O staining using miRNA inhibitor. Western blot assay was performed to examine the expression of IR-related genes and the activation of signaling pathways. A Luciferase experiment was applied to confirm how let-7b-5p regulated the expression of RNF20. IP/WB analysis further determined whether RNF20 promoted STAT3 ubiquitination. Rescue experiment using RNF20 overexpression plasmid was performed to confirm the role of RNF20 on IR-mediated using PC cell-derived exosomes in C2C12 myotube cells. miRNA-let-7b-5p was identified as the key exosomal miRNA, which could promote the IR in C2C12 myotube cells supported the lipid accumulation, the activation of STAT3/FOXO1 axis, and the decreased expression of IRS-1 and GLUT4. RNF20, an E3 ubiquitin ligase, was confirmed as the target gene of let-7b-5p and was found to improve IR by downregulating STAT3 protein expression via ubiquitination-mediated protein degradation. The ectopic expression of RNF20 could effectively attenuate the IR mediated by the pancreatic cancer-derived exosomes in C2C12 myotube cells. Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting RNF20 to activate the STAT3/FOXO1 axis.

MDCT appearances of pancreatic insulinoma as well as the other well differentiated endocrine tumors

Introduction: Pancreatic endocrine tumors (PETs) are primarily well-differentiated tumors composed of cells that resemble normal islet cells but that arise from pancreatic ductal cells. They are classified as functioning or nonfunctioning according to their associated clinical symptoms; insulinomas, gastrinomas and glucagonomas are the most common function­ing PETs Case Report: A 36-years old man presented with a one-month history of fainting attacks and dizziness, symptoms of hypoglycemia such as hunger, sweating, palpitations, loss of consciousness and syncope. We tried abdominal enhanced spiral CT to localize the tumor, but that wasn’t successful. Therefore, we tried a new method of MD spiral CT scanning and localized the tumor. Discussion and Conclusion: Insulinomas are the commonest islet cell tumors of the pancreas, followed by gastrinomas. Preoperative localization is very helpful in planning the operation: it allows the surgeon to determine whether simple tumor resection or partial pancreatectomy is likely to be required.

The role of autophagy in the treatment of type II diabetes and its complications: a review.

Type II diabetes mellitus (T2DM) is a chronic metabolic disease characterized by prolonged hyperglycemia and insulin resistance (IR). Its incidence is increasing annually, posing a significant threat to human life and health. Consequently, there is an urgent requirement to discover effective drugs and investigate the pathogenesis of T2DM. Autophagy plays a crucial role in maintaining normal islet structure. However, in a state of high glucose, autophagy is inhibited, resulting in impaired islet function, insulin resistance, and complications. Studies have shown that modulating autophagy through activation or inhibition can have a positive impact on the treatment of T2DM and its complications. However, it is important to note that the specific regulatory mechanisms vary depending on the target organ. This review explores the role of autophagy in the pathogenesis of T2DM, taking into account both genetic and external factors. It also provides a summary of reported chemical drugs and traditional Chinese medicine that target the autophagic pathway for the treatment of T2DM and its complications.

Identifying target ion channel-related genes to construct a diagnosis model for insulinoma.

Background: Insulinoma is the most common functional pancreatic neuroendocrine tumor (PNET) with abnormal insulin hypersecretion. The etiopathogenesis of insulinoma remains indefinable. Based on multiple bioinformatics methods and machine learning algorithms, this study proposed exploring the molecular mechanism from ion channel-related genes to establish a genetic diagnosis model for insulinoma. Methods: The mRNA expression profile dataset of GSE73338 was applied to the analysis, which contains 17 insulinoma samples, 63 nonfunctional PNET (NFPNET) samples, and four normal islet samples. Differently expressed ion channel-related genes (DEICRGs) enrichment analyses were performed. We utilized the protein-protein interaction (PPI) analysis and machine learning of LASSO and support vector machine-recursive feature elimination (SVM-RFE) to identify the target genes. Based on these target genes, a nomogram diagnostic model was constructed and verified by a receiver operating characteristic (ROC) curve. Moreover, immune infiltration analysis, single-gene gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were executed. Finally, a drug-gene interaction network was constructed. Results: We identified 29 DEICRGs, and enrichment analyses indicated they were primarily enriched in ion transport, cellular ion homeostasis, pancreatic secretion, and lysosome. Moreover, the PPI network and machine learning recognized three target genes (MCOLN1, ATP6V0E1, and ATP4A). Based on these target genes, we constructed an efficiently predictable diagnosis model for identifying insulinomas with a nomogram and validated it with the ROC curve (AUC = 0.801, 95% CI 0.674-0.898). Then, single-gene GSEA analysis revealed that these target genes had a significantly positive correlation with insulin secretion and lysosome. In contrast, the TGF-beta signaling pathway was negatively associated with them. Furthermore, statistically significant discrepancies in immune infiltration were revealed. Conclusion: We identified three ion channel-related genes and constructed an efficiently predictable diagnosis model to offer a novel approach for diagnosing insulinoma.

Establishing novel diagnostic criteria for the glucose tolerance test for the diagnosis of gestational diabetes and gestational hyperglycemia.

To establish diagnostic criteria for the 75-g 2-h glucose tolerance test (GTT) to diagnose gestational diabetes and define the clinical entity of gestational hyperglycemia. A retrospective analysis was performed of the results from 500 patients who had a 75-g 1-h glucose challenge test (GCT) in early pregnancy as part of a two-step approach to screening and testing for gestational diabetes. The selected cohort was considered to have normal islet β-cell function, and upper glycemic levels of normal glucose tolerance in the third trimester were statistically calculated, taking the cutoff threshold values to be the diagnostic criteria for the 75-g 2-h GTT. Gestational hyperglycemia was diagnosed from the false-positive GCT result when ≥8.0 mmol/L (144 mg/dL). The diagnostic criteria for the 75-g 2-h GTT were calculated as follows: fasting plasma glucose ≥5.4 mmol/L (97 mg/dL); 1-h plasma glucose ≥10.5 mmol/L (189 mg/dL); and 2-h plasma glucose ≥8.4 mmol/L (151 mg/dL). The new criteria confirmed a prevalence of gestational diabetes of 11.1% and gestational hyperglycemia of 13.6% in the study population. Novel diagnostic criteria for the 75-g 2-h GTT were established by statistical analysis. This resulted in a more acceptable prevalence of gestational diabetes in our community and the false-positive GCT allowed the detection of gestational hyperglycemia.

Bee Honey Extract Attenuates Hyperglycemia in Induced Type 1 Diabetes: Impact of Antioxidant and Angiogenesis Activities on Diabetic Severity In Vivo

Background: Diabetes mellitus (DM) has become a disease prevalent worldwide. Honey, which comprises predominantly bioactive constituents, has anti-inflammatory, antioxidant, and immunomodulating properties. Aim: Recent developments and benefits of natural products in treating various diseases have caught the attention of researchers. This study aims to investigate the antidiabetic effect of bee honey extract on induced diabetic Swiss mice. Materials and Methods: Fifty Swiss male mice were randomly assigned to five groups of 10 mice each. Group I served as the negative control; in group II, the mice received 2 mg/kg/b.wt of honey extract only; and groups III, IV, and V received cyclosporine (CsA) (20 mg/kg/day, s.c.) daily for 10 days prior to receiving streptozotocin (STZ) inoculated at multiple low doses (MLDSTZ) (30 mg/kg/day, i.p.) for five consecutive days. Group IV was administered with insulin initiated at a dose of 0.5 U/kg/b.wt as a standard treatment (positive control). Group V was administered 2 mg/kg/b.wt of honey extract, while group III received no treatment. Results: The results showed a significant hypoglycemic effect, increased body weight, increased liver glycogen levels, and the amelioration of antioxidant activities in groups IV and V compared with the diabetic group III. Moreover, serum matrix metalloproteinase (MMP-9) concentrations were significantly reduced in the mice treated with the insulin and honey extract in groups IV and V and the tissue inhibitor metalloproteinase-1 (TIMP-1) levels were significantly higher than the serum levels in group III. Furthermore, the histopathological examination of groups IV and V revealed regenerative changes with the restoration of normal islet cell architecture, as compared to the diabetic mice in group III. Compared to group I, group II showed no changes and exhibited non-significant data. Conclusion: Honey extract plays an effective role in improving all biomarkers in treated group V. Furthermore, MMP-9 and TIMP-1 are considered prognostic markers in the progression, severity, diagnosis, and treatment of type 1 DM. This may play an important role for the treatment of individuals in the future.

Correction: Heterogeneous expression of CFTR in insulin-secreting β-cells of the normal human islet

Correction: Heterogeneous expression of CFTR in insulin-secreting β-cells of the normal human islet