Beta-cell Genes Research Articles

Effect of transcription factor MEOX on insulin gene expression in glucagon-like peptide 1-secreting cells.

Currently, the supply of beta cells for islet transplantation in the treatment of type 1 diabetes is limited. Enteroendocrine cells (EECs) are believed to have high potential as stem cells because they share significant developmental similarities with beta cells. In a previous study, we derived EEC cells that secrete individual gut hormones from STC-1 cells. This study aimed to examine intestinal hormone secretion and expression, investigate the expression of developmental-related transcription factors, and analyze the effect of MEOX on insulin gene expression in isolated EECs. The expression and secretion of enteroendocrine hormones were evaluated in L6 and K34 cells from STC-1 cells. Expression patterns of beta cell- and development-related genes in L6 and K34 cells were compared with beta cells. Comparisons of the MEOX-induced expression of Ins in beta cells and GLP-1-secreting cells were investigated. Both L6 and K34 cells predominantly expressed Glp1 and Gip, respectively. The secretion pattern of GLP-1 in L6 cells was similar to that of GLUTag cells. Previous microarray analysis confirmed MEOX as developmentally relevant transcription factors expressed in beta cells. Overexpression of MEOX showed a tendency to increase Ins expression in L6 and GLUTag cells, but not in MIN6 cells. However, when PDX1 and MEOX were co-expressed in GLUTag cells, insulin expression was suppressed, similar to that observed in MIN6 cells. These findings suggest a potential role for MEOX in regulating the expression of the Ins gene in both beta cells and GLP-1-secreting cells. Further studies are warranted to elucidate the underlying mechanism.

SAT092 A Case Of Mature Onset Diabetes Of The Young

Abstract Disclosure: L. Beshay: None. A. Thakkar: None. Background: Maturity onset diabetes of the young (MODY) is a disorder characterized by noninsulin-dependent diabetes diagnosed at a young age (<25 years) with autosomal dominant inheritance and lack of autoantibodies. It accounts for 2 to 5 percent of diabetes cases. Several different genetic abnormalities have been identified causing disruption in pancreatic beta cells development and function leading to impaired insulin secretion. Mutations in hepatocyte nuclear factor-1-alpha (HNF1A) is the most commonly identified mutation occurring in 52 to 65 of MODY cases. Case Description: A 21 year old female with a history of diabetes and recurrent skin abscesses presented to the emergency room with right lower quadrant epidermal abscess. Endocrinology was consulted for evaluation of her diabetes. She was diagnosed at age 14 when she presented with weight loss, polyuria, polydipsia, BG 406 without diabetic ketoacidosis. She reported a history of type 2 diabetes in mother. Her medications included metformin, lantus and humalog. It was noted that her diabetes was never controlled with an A1c persistently >10%. Patient reported skin swelling with insulin injections and working two jobs which limited her ability to take more than one injection per day. Her BMI is 23 kg/m2. Labs this admission showed HbA1c 13.7%, GAD 65 Ab and IA-2 Ab negative, c-peptide 1.1 ng/ml, with a glucose of 209 mg/dl. MODY was suspected due to her age, intact c-peptide, and lack of DM1 antibodies. The patient was discharged on Trulicity 0.75 mg weekly, Lantus 12U nightly, Glipizide 2.5mg daily. On follow up, a MODY genetic panel confirmed a HNF1A missense variant with AD inheritance. Since initiation of sulfonylurea, she has had improved control of diabetes. Glipizide dose is being titrated up with concomitant decrease in insulin dose with plan to discontinue basal insulin once her blood sugars are at goal. Conclusion: The HNF1A gene on chromosome 12 is a weak transactivator of the insulin gene in beta cells. Mutations of HNF1A can lead to abnormal insulin secretion and a low renal threshold for glucose. These patients are at risk for micro- and macrovascular complications of diabetes. In terms of treatment, they exhibit increased insulin sensitivity and marked sensitivity to the hypoglycemic effects of sulfonylureas compared with patients with type 2 diabetes. Thus, patients can be successfully treated with sulfonylurea monotherapy, and in one clinical study, approximately 70 percent of patients previously treated with insulin successfully switched to sulfonylureas. Thus, it is important to evaluate etiology of diabetes, as detection of MODY variants in which sulfonylureas are used for treatment simplifies management and improves outcomes as demonstrated in this case. Presentation: Saturday, June 17, 2023

Inferring regulators of cell identity in the human adult pancreas.

Cellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity and cell states in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. BHLHE41 depletion induced apoptosis in primary pancreatic islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity and cell states in the human adult pancreas.

Beta cell dysfunction induced by bone morphogenetic protein (BMP)-2 is associated with histone modifications and decreased NeuroD1 chromatin binding

SummaryInsufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

135-OR: Mapping Cis-Regulatory Programs Affecting Diabetes Risk in Pancreatic Islet Cell Types Using Single-Cell Multimodal Profiling

Genetic variants influencing type 1 diabetes (T1D) risk identified by genome-wide association studies (GWAS) are primarily non-coding and affect cis-regulatory element (cRE) activity in specific cell types. A more detailed understanding of cell type-specific cis-regulatory programs in the pancreas is therefore crucial for deciphering the functional impact of T1D risk. Recently developed assays that generate multimodal single nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) profiles from the same cell enable high-resolution mapping of cis-regulatory programs in specific cell types. We generated deeply sequenced data from 28 nondiabetic pancreatic islet samples from the Alberta Diabetes Institute IsletCore using 10x multiome assays. After extensive quality control and filtering, we clustered multimodal profiles from 174,819 cells and identified ten major cell types. For several cell types we identified evidence of heterogeneous sub-populations; for example, in beta cells there were six sub-populations corresponding to cellular states associated with insulin secretion, stress response, and others. We identified 263,223 cREs across all cell types and developed a novel method to predict target genes of cREs based on the correlation of multimodal profiles across single cells. In beta cells, there were 122,352 cRE-target gene links involving 16,907 unique genes. We mapped chromatin accessibility quantitative trait loci (caQTLs) for cREs in each cell type and identified a total of 14,535 caQTLs at FDR<.10. Finally, we annotated T1D risk variants from high-resolution fine-mapping of 136 T1D signals with cRE-target gene links and caQTLs. At 27 T1D signals including at the INS, MAPT, and DLK1 loci, variants affecting chromatin accessibility were linked to putative target genes in beta cells suggesting mechanisms of action at these loci. Experimental follow up of variant activity at these loci using genome editing in beta cell lines is ongoing. Disclosure H. Mummey: None. W. Elison: None. K. Korgaonkar: None. Y.S. Lee: None. J.M. Newsome Ashmus: None. P. Benaglio: Employee; Shoreline Biosciences. M.T. Miller: None. J.E. Manning Fox: None. A.L. Gloyn: Other Relationship; Genentech, Inc., Roche Pharmaceuticals. P. MacDonald: None. S. Preissl: None. K.J. Gaulton: Stock/Shareholder; Neurocrine Biosciences, Inc. Consultant; Genentech, Inc. Stock/Shareholder; Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (HG012059, DK105554, DK122607, DK114650)

1773-P: Glucocorticoid Receptor Signaling Regulates Pancreatic Beta-Cell Circadian Transcriptome and Function

Glucocorticoid (GC) signaling is an important regulator of glucose homeostasis. GC secretion also serves as an entrainment signal that coordinates circadian gene expression of peripheral circadian clocks. Pancreatic beta cells are key biological controllers of glucose homeostasis through the coordinated circadian release of insulin. However, the role of GC signaling in the regulation of beta cell circadian clocks and insulin secretion remains unknown. To address this, we first performed chromatin immunoprecipitation/sequencing (ChIP-seq) in beta cell lines (MIN6 and INS1) and identified >1,000 glucocorticoid receptor (GR)-bound regions (FDR<0.01) in response to the synthetic GC dexamethasone (DEX, 100nM). Annotation of GR-bound regions showed enrichment for pathways regulating insulin secretion and circadian rhythms. To further explore the effects of GC on circadian beta cell gene expression and function in vivo, we used C57BL/6 mice housed under either 12h/12h light-dark cycles (LD) or constant light-mediated circadian disruption (CD). Mice were subsequently administered daily intraperitoneal injections of DEX (2 mg/kg) or vehicle, designed to restore circadian secretion of GC disrupted in CD group. DEX treatment normalized circadian regulation of glucose tolerance (p<0.05 vs. vehicle) and glucose-stimulated insulin secretion (p<0.05 vs vehicle) in CD group. We next utilized RNA sequencing to assess circadian transcriptional profiles in islets isolated from LD, CD, and CD + DEX mice. Consistent with previous studies, CD resulted in abrogation of circadian islet mRNA expression, whereas DEX restored circadian rhythmicity and enhanced expression of core clock genes (e.g., Bmal1, Dbp, Nr1d1) and genes regulating beta cell identity (e.g., Ins1, Nkx6.1) and insulin secretion (e.g., Gck, Irs2). These data highlight the GC/GR axis as a direct regulator of beta cell circadian gene expression, insulin secretion and glucose tolerance. Disclosure S.K.Sen: None. L.Wang: None. M.Brown: None. J.Wu: None. Z.Wei: None. A.Matveyenko: None. Funding National Institutes of Health (DK098468)

349-OR: Selected DYRK1A Inhibitors Simultaneously Drive Both Human Beta-Cell Proliferation and Differentiation

Small molecule DYRK1A inhibitors induce adult human beta cell proliferation and expand human beta cell mass. Harmine also enhances differentiation and function in human beta cells in vitro and in vivo, increasing expression of key beta cell genes. It is unknown whether this “pro-differentiation effect” is a feature of all DYRK1A inhibitors. We compared harmine, 2-2c, 5-Iodotubericidin (5-IT), GNF4877, INDY, CC-401 and Leucettine-41 at doses that maximally drive human beta cell proliferation. As anticipated, harmine, 2-2c and 5-IT all increased expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC30A8, and ENTPD3. These effects were observed in normal human islets as well as in islets from donors with T2D. Surprisingly, in simultaneous experiments, the DYRK1A inhibitors GNF4877, INDY, CC-401 and Leucettine failed to induce these beta cell differentiation markers. The pro-differentiation effect of harmine was independent of DYRK1A inhibition: silencing DYRK1A in human islets induced beta cell proliferation, but had no effect on differentiation; harmine treatment in DYRK1A-silenced islets restored beta cell pro-differentiation effects. The pro-differentiation effect was also independent of NFAT signaling and DREAM Complex effects, since NFAT overexpression, or p130, p107, E2F4 and E2F5 silencing had no effect on beta cell differentiation. Similarly, silencing other known harmine targets (DYRK2, 3, 4, the CLK and HIPK family) had no effect on either human beta cell differentiation nor proliferation. In summary, harmine, 2-2c and 5-IT are unique among the DYRK1A inhibitor family in their simultaneous ability to enhance both beta cell proliferation and differentiation. While the beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor drug development. Disclosure P. Wang: None. K. Kumar: None. H. Liu: None. O. Wood: None. E. Karakose: None. L. Choleva: None. L. Lambertini: None. A. Garcia-Ocana: Consultant; Sun Pharmaceutical Industries Ltd. R.J. DeVita: None. A.F. Stewart: None.

A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells.

Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.

A Case of Hyperinsulinemic Hypoglycemia in Premature Infant Treated with Oral Diazoxide

Neonatal hyperinsulinism, whether permanent or transient, results in prolonged hypoglycemia, which increases the risk of hypoglycemic brain injury. Therefore, prompt diagnosis and management of hyperinsulinemic hypoglycemia is important. Drawing a “critical sample” at the time of hypoglycemia is useful for diagnosis. Genetic testing for defective insulin-regulating genes in pancreatic beta-cells might also be helpful in cases of prolonged hypoglycemia. High-calorie feeding or glucose infusion is necessary to maintain normoglycemia. Diazoxide is the treatment of choice for hyperinsulinism and should be continued until the hypoglycemia resolves. We describe a case of transient neonatal hyperinsulinemia hypoglycemia in a small-for-gestational-age preterm infant who underwent diazoxide treatment and achieved a favorable outcome.

Abstract C050: Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Various epidemiologic studies have shown that obesity increases the risk of PDAC. With rising rates of obesity worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. Therefore, to study the effect of obesity on PDAC, we combined a well-established genetic model of obesity (Lepob/ob) with a faithful oncogenic Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) and showed that obese mice had both increased tumor burden and shortened survival compared to their lean counterparts. Strikingly, weight loss (via AAV-leptin rescue or caloric restriction), decreased tumor development, demonstrating that the tumor-promoting effects of obesity can be reversed. Bulk-RNA sequencing of tumors from obese mice exhibited aberrant expression of islet beta-cell genes, including those coding for the peptide hormones cholecystokinin (CCK) and amylin (Iapp). Single-cell RNA sequencing of islet beta-cells from wild-type, high fat diet-fed, and Lepob/ob mice revealed a trajectory of beta-cell adaptation to obesity with dysregulated hormone expression, including downregulation of insulin expression and upregulation of CCK and Iapp, concordant with loss of beta-cell identity transcription factors and beta-cell dysfunction. In silico analyses using dynamic optimal transport (TrajectoryNet) demonstrate that CCK-expressing beta-cells arise from pre-existing insulin low-expressing beta-cells, arguing for baseline beta-cell heterogeneity as a determinant of aberrant hormone expression. Lastly, transgenic expression of CCK or Iapp in beta-cells was sufficient to promote oncogenic Kras-driven pancreatic tumorigenesis, while converse beta-cell ablation impeded tumor progression in mice. Together, these results uncover a novel mechanism of obesity driven PDAC by local hormonal signaling between endocrine islets and exocrine acinar cells and reveal pancreatic endocrine adaptations that could be targeted to halt exocrine tumorigenesis. Citation Format: Cathy C. Garcia, Lauren Lawres, Sherry Agabiti, Jaffarguriqbal Singh, Alex Tong, Aarthi Venkat, Daniel B. Burkhardt, Rebecca Cardone, Richard G. Kibbey, Smita Krishnaswamy, Mandar D. Muzumdar. Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C050.

Proteasomal degradation of WT proinsulin in pancreatic beta cells

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum–associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis.

296-OR: Single-Cell Multiomic Analysis of the Human Pancreas in Type 1 Diabetes

The cis-regulatory programs of specific cell types in the pancreas that are affected in type 1 diabetes (T1D) are poorly understood. Single cell technology enables detailed interrogation of genomic profiles of cell types within a heterogeneous tissue, and when applied to samples across disease states can reveal cell type-specific changes in disease. In this study we applied single nucleus RNA-seq (snRNA-seq) and single nucleus ATAC-seq (snATAC-seq) , as well as paired multiome (snRNA-seq+snATAC-seq) , to cryopreserved whole pancreas samples from 34 donors including 13 control, 9 T1D autoantibody positive (Aab+) , 7 early T1D (1< yr) and 5 late T1D donors in the nPOD biorepository. Clustering of 313,078 gene expression and 342,863 accessible chromatin profiles after extensive processing and filtering resolved pancreatic endocrine, exocrine, immune, stellate, glial and endothelial cell types, as well as known and novel cell sub-types for example REG1A+ acinar cells and MUC5b+ ductal cells. There were marked changes in the abundance of many cell types and sub-types across disease states, including a significant reduction in beta cells and increase in immune cell populations in T1D compared to control. Differential analysis of gene expression and cis-regulatory element (cCRE) activity in each cell type revealed changes in early and late T1D, as well as in T1D Aab+. In beta cells, 2 and 2,177 genes and 1,080 and 9,177 cCREs had differential activity in early and late T1D, respectively, and early and late T1D genes in beta cells were enriched for distinct molecular processes related to interferon gamma signaling and cell death. Intersecting differential beta cell cCREs with comprehensive T1D genetic fine-mapping revealed 13 T1D signals where candidate variants mapped in a differential beta cell cRE of which were proximal to a differentially expressed gene, such as at the GLIS3 locus. Together these results provide insight into the role of cis-regulatory programs in beta cells and other pancreatic cell types in T1D. Disclosure R.Melton: None. S.Preissl: None. M.Sander: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. E.Beebe: None. C.Zeng: None. M.T.Miller: None. G.Wang: None. C.Mcgrail: None. H.Mummey: None. I.Kusmartseva: None. M.A.Atkinson: None. Funding Ruth Kirschstein Institutional National Research Service Award T32 (GM008666) National Institutes of Health (DK120429)

Comprehensive Analysis of Ubiquitously Expressed Genes in Humans from A Data-driven Perspective

Comprehensive characterization of spatial and temporal gene expression patterns in humans is critical for uncovering the regulatory codes of the human genome and understanding the molecular mechanisms of human diseases. Ubiquitously expressed genes (UEGs) refer to the genes expressed across a majority of, if not all, phenotypic and physiological conditions of an organism. It is known that many human genes are broadly expressed across tissues. However, most previous UEG studies have only focused on providing a list of UEGs without capturing their global expression patterns, thus limiting the potential use of UEG information. In this study, we proposed a novel data-driven framework to leverage the extensive collection of ∼ 40,000 human transcriptomes to derive a list of UEGs and their corresponding global expression patterns, which offers a valuable resource to further characterize human transcriptome. Our results suggest that about half (12,234; 49.01%) of the human genes are expressed in at least 80% of human transcriptomes, and the median size of the human transcriptome is 16,342 genes (65.44%). Through gene clustering, we identified a set of UEGs, named LoVarUEGs, which have stable expression across human transcriptomes and can be used as internal reference genes for expression measurement. To further demonstrate the usefulness of this resource, we evaluated the global expression patterns for 16 previously predicted disallowed genes in islet beta cells and found that seven of these genes showed relatively more varied expression patterns, suggesting that the repression of these genes may not be unique to islet beta cells.

Evidence for a Highly Cytokine‐Sensitive Network of Iron‐Associated Genes in Islets

Among the complex factors contributing to beta‐cell failure is inflammation. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)‐1beta and IL‐6, increases the risk of developing type‐2 diabetes (T2D), and there is evidence that these low levels of circulating cytokines lead to islet dysfunction. Iron is closely connected to both the inflammatory response and diabetes. High levels of dietary iron increase risk of developing T2D, and excessive iron uptake by beta‐cells can cause oxidative stress and inhibit function. In this study, islets were treated for 48h with 10 pg/mL IL‐1beta + 20 pg/mL IL‐6 as a model of low‐grade inflammation. Analysis of gene microarray data identified three iron‐associated genes among the most cytokine‐sensitive: HAMP, STEAP4, and LCN2. These proteins are all involved directly or indirectly with increasing and/or sequestering cellular iron. RT‐PCR following exposure to various stressors known to induce beta‐cell failure revealed upregulation of HAMP, STEAP4, and LCN2 to be cytokine‐specific. Overexpression of STEAP4 induced upregulation of HAMP and LCN2, while ferritin and ferroportin expression were unaffected. Beta‐cells scanned by synchrotron X‐Ray fluorescence provided data showing cytokine exposure alters iron distribution. Iron was found in discrete structures (area ~0.15‐0.45 µm2) that were significantly smaller and more iron‐dense in cytokine‐treated beta‐cells, consistent with the sequestering function of identified iron‐associated genes. These data suggest a network of iron‐regulating genes in beta‐cells plays a role in sequestering iron in response to low‐grade inflammation.

Dual regulation of miR-375 and CREM genes in pancreatic beta cells

ABSTRACT MicroRNA-375 (miR-375) is upregulated in the islets of some diabetics and is correlated with poor outcome. Previous work in our laboratory showed that cyclic adenosine monophosphate (cAMP) reduces miR-375 expression and could provide a way to restore normal miR-375 levels, however the transcription repression mechanism is unknown. Using a chromatin immunoprecipitation assay we show that cAMP response element modulator (CREM) binds to the miR-375 promoter 3-fold above background and we find that CREM represses transcription from the miR-375 promoter 1.8-fold. While investigating miR-375 target genes we discovered that several microRNA:mRNA target prediction algorithms listed human CREM as a target gene of miR-375. The predicted binding site is conserved in primates but not in other species. We found that indeed miR-375 binds to the predicted site on human CREM and represses translation of a green fluorescent protein reporter gene by 30%. These findings suggest a primate-specific double-negative feedback loop, a mechanism that would keep these important β-cell regulators in check.

Role of monogenic diabetes genes on beta cell function in Italian patients with newly diagnosed type 2 diabetes. The Verona Newly Diagnosed Type 2 Diabetes Study (VNDS) 13

We tested the hypothesis that common genetic variability of beta-cell genes responsible for monogenic diabetes may affect beta cell function in type 2 diabetes mellitus (T2DM). We studied 794 drug- naïve GAD-negative patients with newly diagnosed T2DM (age: median=59 years; I.Q. range: 52-66; body mass index: 29.3 kg/m2; 26.6-32.9). Beta-cell function was assessed by state-of-art mathematical modeling of glucose/C-peptide curves during a 240'-300' frequently sampled oral glucose tolerance test, to provide the beta-cell responses to the rate of increase in glucose concentration (derivative control: DC) and to glucose concentration (proportional control: PC). Forty-two single nucleotide polymorphism (SNPs), selected to cover over 90% of common genetic variability, were genotyped in nine monogenic diabetes genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, KCNJ11 and ABCC8. Allelic variants of four SNPs (rs1303722 and rs882019 of GCK, rs7310409 of HNF1A and rs5219 of KCNJ11) were significantly associated with DC of beta-cell secretion (all P < 0.036). Allelic variants of four other SNPs (rs2868094 and rs6031544 of HNF4A, and rs1801262 and rs12053195 of NEUROD1) were associated with PC of beta-cell secretion (P < 0.02). In multivariate models, GCK, HNF1A and KCNJ11 SNPs explained 2.5% of the DC variability of beta-cell secretion, whereas HNF4A and NEUROD1 SNPs explained 3.6% of the PC variability of beta-cell secretion. We conclude that common variability of monogenic diabetes genes is significantly associated with an impaired beta-cell function in patients with newly diagnosed T2DM; thereby, these genes might be targeted by specific treatments in T2DM.

The Role of Fatty Acid Signaling in Islet Beta-Cell Adaptation to Normal Pregnancy

BackgroundMaintenance of a normal fetal nutrient supply requires major adaptations in maternal metabolic physiology, including of the islet beta-cell. The role of lipid signaling processes in the mechanisms of islet beta-cell adaptation to pregnancy has been minimally investigated.ObjectiveTo determine the effects of pregnancy on islet fatty acid (FA) metabolic partitioning and FA augmentation of glucose-stimulated insulin secretion (GSIS).MethodsAge matched virgin, early pregnant (gestational day-11, G11) and late pregnant (G19) Sprague-Dawley rats were studied. Fasted and fed state biochemistry, oral glucose tolerance tests (OGTT), and fasted and post-OGTT liver glycogen, were determined to assess in vivo metabolic characteristics. In isolated islets, FA (BSA-bound palmitate 0.25 mmol/l) augmentation of GSIS, FA partitioning into esterification and oxidation processes using metabolic tracer techniques, lipolysis by glycerol release, triacylglycerols (TG) content, and the expression of key beta-cell genes were determined.ResultsPlasma glucose in pregnancy was lower, including during the OGTT (glucose area under the curve 0-120 min (AUC0-120); 655±24 versus 849±13 mmol.l-1.min; G19 vs virgin; P<0.0001), with plasma insulin concentrations equivalent to those of virgin rats (insulin AUC0-120; 97±7 versus 83±7 ng.ml-1.min; G19 vs virgin; not significant). Liver glycogen was depleted in fasted G19 rats with full recovery after oral glucose. Serum TG increased during pregnancy (4.4±0.4, 6.7±0.5; 17.1±1.5 mmol/l; virgin, G11, G19, P<0.0001), and islet TG content decreased (147±42, 172±27, 73±13 ng/µg protein; virgin, G11, G19; P<0.01). GSIS in isolated islets was increased in G19 compared to virgin rats, and this effect was augmented in the presence of FA. FA esterification into phospholipids, monoacylglycerols and TG were increased, whereas FA oxidation was reduced, in islets of pregnant compared to virgin rats, with variable effects on lipolysis dependent on gestational age. Expression of Ppargc1a, a key regulator of mitochondrial metabolism, was reduced by 51% in G11 and 64% in G19 pregnant rat islets compared to virgin rat islets (P<0.001).ConclusionA lowered set-point for islet and hepatic glucose homeostasis in the pregnant rat has been confirmed. Islet adaptation to pregnancy includes increased FA esterification, reduced FA oxidation, and enhanced FA augmentation of glucose-stimulated insulin secretion.

Insulin is expressed by enteroendocrine cells during human fetal development.

Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here we reconstruct a single-cell atlas of the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and expose its molecular blueprint.

A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis

ABSTRACT Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression. We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples. We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokines – or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

Benefits of Sustained Upregulated Unimolecular GLP-1 and CCK Receptor Signalling in Obesity-Diabetes

Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys12Pal]Ex-4/CCK. Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.