Mouse Pancreatic Cells Research Articles

7880 The Role Of PTHrP In Glucose Metabolism And Insulin Secretion

Abstract Disclosure: I.M. Max-Harry: None. N. Kantake: None. C.S. Nunemaker: None. T.J. Rosol: None. Insulin and glucagon are the two most important hormones for blood glucose homeostasis. Identifying the key players in their regulation will contribute greatly towards combatting types 1 and 2 diabetes. Parathyroid Hormone-related protein (PTHrP), a protein expressed by pancreatic beta cells, has been implicated in various developmental and physiological roles, but particularly, PTHrP increases beta-cell proliferation and insulin secretion in pancreatic islets. Incubating mouse pancreatic islets and Min6 cells with 10nM exogenous PTHrP showed a significant increase in both insulin secretion (for islets, PTHrP: 865.4ng/ml ± 77.66, con: 498.5ng/ml ± 40.63, p=0.024, n=4) and glucagon secretion (PTHrP: 20.87pmol/l ± 3.776, con: 8.798pmol/l ± 1.616, p= 0.068, n=3) after 24hours. In order to further investigate the effects observed, min6 cells were transiently transfected to overexpress PTHrP, and a glucose-stimulated insulin secretion (GSIS) assay was carried out with 2mM and 20mM glucose. The results showed a reduction in basal insulin secretion at 2mM glucose and an increase in insulin secretion at 20mM glucose for the PTHrP transfected cells. PTHrP improved the stimulation index of Min6 cells (PTHrP: 2.279 ± 0.2056, con: 1.659 ± 0.1386, p= 0.03, n=6) and enhanced their sensitivity to glucose. Preliminary proliferation assays showed that cells transfected with PTHrP have increased proliferation and a protection from cell death when incubated in serum-free media. This study, in addition to our previously published research showing that a lack of intact PTHrP leads to islet dysfunction, revealed that PTHrP may be crucial to appropriate glucose sensitivity of beta cells. Further studies are being carried out to understand the mechanism behind these effects and the genes/proteins involved in the regulation of insulin secretion by PTHrP. Presentation: 6/1/2024

M6A mRNA methylation by METTL14 regulates early pancreatic cell differentiation

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported that m6A is involved in the postnatal control of β-cell function in physiological states and in type 1 and 2 diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells.

Multiple promoter and enhancer differences likely contribute to augmented G6PC2 expression in human versus mouse pancreatic islet alpha cells.

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in β-cells than in α-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in β-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in α-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in α-cells versus β-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse β-cells versus α-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between α-cells and β-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function.

Isolation of Mouse Pancreatic Endothelial Cells.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

Therapeutic expression of RAS Degrader RRSP in Pancreatic Cancer via Nanocarrier-mediated mRNA delivery.

About one-third of all human cancers encode abnormal RAS proteins locked in a constitutively activated state to drive malignant transformation and uncontrolled tumor growth. Despite progress in development of small molecules for treatment of mutant KRAS cancers, there is a need for a pan-RAS inhibitor that is effective against all RAS isoforms and variants and that avoids drug resistance. We have previously shown that the naturally occurring bacterial enzyme RAS/RAP1-specific endopeptidase (RRSP) is a potent RAS degrader that can be re-engineered as a biologic therapy to induce regression of colorectal, breast, and pancreatic tumors. Here, we have developed a strategy for in vivo expression of this RAS degrader via mRNA delivery using a synthetic nonviral gene delivery platform composed of the poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) block copolymer conjugated to a dendritic cationic peptide (PPDP2). Using this strategy, PPDP2 is shown to deliver mRNA to both human and mouse pancreatic cells resulting in RRSP gene expression, activity, and loss of cell proliferation. Further, pancreatic tumors are reduced with residual tumors lacking detectable RAS and phosphorylated ERK. These data support that mRNA-loaded synthetic nanocarrier delivery of a RAS degrader can interrupt the RAS signaling system within pancreatic cancer cells while avoiding side effects during therapy.

Selective epigenetic alterations in RNF43 in pancreatic exocrine cells from high-fat-diet-induced obese mice; implications for pancreatic cancer

ObjectivePancreatic cancer (PC) originates and progresses with genetic mutations in various oncogenes and suppressor genes, notably KRAS, CDKN2A, TP53, and SMAD4, prevalent across diverse PC cells. In addition to genetic mutations/deletions, persistent exposure to high-risk factors, including obesity, induces whole-genome scale epigenetic alterations contributing to malignancy. However, the impact of obesity on DNA methylation in the presymptomatic stage, particularly in genes prone to PC mutation, remains uncharacterized.ResultsWe analyzed the methylation levels of 197 loci in six genes (KRAS, CDKN2A, TP53, SMAD4, GNAS and RNF43) using Illumina Mouse Methylation BeadChip array (280 K) data from pancreatic exocrine cells obtained from high-fat-diet (HFD) induced obese mice. Results revealed no significant differences in methylation levels in loci between HFD- and normal-fat-diet (NFD)-fed mice, except for RNF43, a negative regulator of Wnt signaling, which showed hypermethylation in three loci. These findings indicate that, in mouse pancreatic exocrine cells, high-fat dietary obesity induced aberrant DNA methylation in RNF43 but not in other frequently mutated PC-related genes.

Abstract A046: nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. Packaging of specific cellular constituents, such as prion protein and specific miRNAs involved in cancer progression, into exosomes are dependent on nSMase2. Employing the KPC mouse model of pancreatic ductal adenocarcinoma (PDA), we demonstrate that nSMase2 regulates exosome secretion and in vivo growth of PDA cells. PDA cell exosomes generated through nSMase2 accelerate PDA progression, and PDA cell exosomes generated independently of nSMase2 are anti-tumorigenic during PDA progression. Homozygous loss of Smpd3 (gene which encodes nSMase2) in KPC mouse pancreatic epithelial cells during development extends survival of KPC mice. KPC; Smpd3f/f mice display less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. In addition, nSMase2 loss in KPC PDA cells promoted a PDA subtype switch from aggressive basal-like to classical. RNA-sequencing of PDA cell lines generated from pancreatic tumors of end-stage KPC; Smpd3wt/wt, KPC; Smpd3f/wt, and KPC; Smpd3f/f mice demonstrated that nSMase2-generated ceramide is a key regulator of intracellular pathways regulating tumor microenvironment dynamics in PDA cells including fibrosis and integrins. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt mouse pancreata. Exosomes isolated from KPC; Smpd3wt/wt PDA cell lines significantly stimulate proliferation of primary murine stellate cells in vitro, while exosomes isolated from KPC; Smpd3f/f PDA cell lines do not. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive proinflammatory macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Proteomics and small RNA-seq analysis of exosomes secreted from KPC; Smpd3wt/wt and KPC; Smpd3f/f PDA cell lines demonstrated significant alteration of >700 proteins and 12 miRNAs. Our patient data demonstrate that low nSMase2 expression in treatment naïve primary resected pancreatic tumors significantly correlates with fewer CD31+ endothelial cells. In parallel, loss of Smpd3 resulted in a reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt controls. Thus, nSMase2 is a critical mediator of angiogenesis during PDA progression, which is integral to chemotherapeutic response. Collectively, our data show that nSMase2-generated exosomes promote PDA tumorigenesis, specifically proliferation of stellate cells, which may in turn induce a fibrotic tumor microenvironment that affects vasculature development. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Tuan A. Phu, Martin Ng, Natanya R. Kerper, David I. Berrios, Jonghyun Lee, David Jin, Gun H. Jang, Oghenekevwe M. Gbenedio, Jeroen P. Roose, Jen J. Yeh, Steven Gallinger, Andrew V. Biankin, Vasilis Ntranos, David K. Chang, David W. Dawson, Grace E. Kim, Valerie M. Weaver, Robert L. Raffai, Matthias Hebrok. nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A046.

CDN1163, an activator of sarco/endoplasmic reticulum Ca2+ ATPase, up-regulates mitochondrial functions and protects against lipotoxicity in pancreatic β-cells.

High levels of Ca2+ in the endoplasmic reticulum (ER), established by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), are required for protein folding and cell signalling. Excessive ER Ca2+ release or decreased SERCA activity induces unfolded protein accumulation and ER stress in pancreatic β-cells, leading to defective insulin secretion and diabetes. Here we have investigated the consequences of enhancing ER Ca2+ uptake on β-cell survival and function. The effects of SERCA activator, CDN1163, on Ca2+ homeostasis, protein expression, mitochondrial activities, insulin secretion, and lipotoxicity have been studied in mouse pancreatic β-cells and MIN6 cells. CDN1163, increased insulin synthesis and exocytosis from islets. CDN1163 also increased the sensitivity of the cytosolic Ca2+ oscillation response to glucose and potentiated it in dispersed and sorted β-cells. CDN1163 augmented the ER and mitochondrial Ca2+ content, the mitochondrial membrane potential, respiration, and ATP synthesis. CDN1163 up-regulated expression of inositol 1,4,5-trisphosphate receptors and antioxidant enzymes, and mitochondrial biogenesis, including peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). Overexpression of SERCA2a or 2b replicated the effects of CDN1163, while knockdown of SERCA2 abolished the stimulatory actions of CDN1163. In palmitate-treated β-cells, CDN1163 prevented ER Ca2+ depletion, mitochondrial dysfunction, cytosolic and mitochondrial oxidative stress, defective insulin secretion, and apoptotic cell death. Activation of SERCA enhanced mitochondrial bioenergetics and antioxidant capability, suppressing the cytotoxic effects of palmitate. Our results suggest that targeting SERCA could be a novel therapeutic strategy to protect β-cells from lipotoxicity and the development of Type 2 diabetes.

Wfs1 loss-of-function disrupts the composition of mouse pancreatic endocrine cells from birth and impairs Glut2 localization to cytomembrane in pancreatic β cells

Wfs1 is an endoplasmic reticulum (ER) membrane located protein highly expressed in pancreatic β cells and brain. Wfs1 deficiency causes adult pancreatic β cells dysfunction following β cells apoptosis. Previous studies mainly focus on the Wfs1 function in adult mouse pancreatic β cells. However, whether Wfs1 loss-of-function impairs mouse pancreatic β cell from its early development is unknown. In our study, Wfs1 deficiency disrupts the composition of mouse pancreatic endocrine cells from early postnatal day 0 (P0) to 8 weeks old, with decreased percentage of β cells and increased percentage of α and δ cells. Meanwhile, Wfs1 loss-of-function leads to reduced intracellular insulin content. Notably, Wfs1 deficiency impairs Glut2 localization and causes the accumulation of Glut2 in mouse pancreatic β cell cytoplasm. In Wfs1-deficient mice, glucose homeostasis is disturbed from early 3 weeks old to 8 weeks old. This work reveals that Wfs1 is significantly required for the composition of pancreatic endocrine cells and is essential for Glut2 localization in mouse pancreatic β cells.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition

Background and aimsThiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear.MethodsTo model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl– and HCO3– secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization.ResultsWe demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3– and Cl– secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR.ConclusionsAZA impaired the ductal HCO3– and Cl– secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

MicroRNA-320–3p promotes the progression of acute pancreatitis by blocking DNMT3a-mediated MMP8 methylation in a targeted manner

In this research, we screened out two genes upregulated in mice with acute pancreatitis (AP) by gene sequencing: microRNA (miR)-320–3p and matrix metalloprotease 8 (MMP8). This study was designed to determine whether miR-320–3p and MMP8 participate in AP development and explore the mechanisms, with a new idea for clinical diagnosis and treatment of AP. Expression of miR-320–3p, DNA methyltransferase 3a (DNMT3a), and MMP8 in mouse pancreatic tissues and AR42J cells was tested by RT-qPCR and western blot assays. Pancreatic pathological changes, serum amylase and lipase, and inflammatory factors in mouse serum and cell supernatant were measured by hematoxylin-eosin staining, automation analyzer, and enzyme-linked immunosorbent assay, respectively. Cell proliferation and apoptosis were determined by CCK-8 assay and flow cytometry. The interaction between miR-320–3p, DNMT3a, and MMP8 was verified by luciferase activity assay, ChIP-qPCR, and MSP assay. High expression of miR-320–3p and MMP8, and low expression of DNMT3a were observed in pancreatic tissues of AP mice and caerulein-induced AP cellular model. Downregulation of miR-320–3p alleviated injury of mouse pancreas, reduced the levels of serum amylase and lipase, and blocked inflammatory factor levels in AP mice. In caerulein-induced AP cellular models, inhibiting miR-320–3p facilitated proliferation and inhibited apoptosis. Upregulation of MMP8 resulted in the opposite results, which could be reversed by simultaneous inhibition of miR-320–3p. miR-320–3p targeted DNMT3a, and downregulating miR-320–3p promoted DNMT3a expression. Moreover, DNMT3a promoted DNA methylation in MMP8 promoter region, thereby inhibiting MMP8 expression in AP mouse and cellular models. This research suggests that miR-320–3p inhibits DNMT3a to reduce MMP8 methylation and increase MMP8 expression, thereby promoting AP progression.

Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection

An efficient transfection is a crucial step for the introduction of epigenetic modification in host cells, and there is a need for an optimized transfection process for individual model systems separately. Mouse pancreatic αTC1-6 cells, which act as an attractive model system for epigenetic cell reprogramming and diabetes treatment, were transiently transfected with two different transfection methods: the chemical method with polyethyleneimine (PEI) and nucleofection as a physical transfection method. Flow cytometry and fluorescent microscopy examination of GFP expression showed that transfection efficiency was affected by the size of plasmids using both transfection methods. Subsequently, the Cas9 mRNA expression confirmed successful transfection with EpiCRISPR plasmid, whereas the cell physiology remained unchanged. The adjusted nucleofection protocol for αTC1-6 cells transfected with an EpiCRISPR mix of plasmids reached 71.1% of GFP-positive transfected cells on the fifth post-transfection day and proved to be much more efficient than the 3.8% GFP-positive PEI transfected cells. Modifying the protocol, we finally specify CM-156 program and SF 4D-Nucleofector X Solutions for Amaxa™ nucleofection as a method of choice for alpha TC1-6 cell line transfection.

Combined Inhibition of FOSL-1 and YAP Using siRNA-Lipoplexes Reduces the Growth of Pancreatic Tumor.

Simple SummaryIntercepting the molecular mechanisms implicated in pancreatic cancer progression can be an efficient therapeutic approach to treat this aggressive tumor. The Hippo pathway is a key mechanism driving tumor progression, even in the absence of KRAS activation. When this pathway is switched off, the transcriptional coactivator YAP is translocated into the nucleus and induces the activation of several genes implicated in tumor progression and apoptosis inhibition. FOSL-1 is a transcription factor that synergizes with YAP, forming a transcriptional complex. It has been shown to have a good therapeutic outcome when they are individually inhibited. In this work, we showed for the first time that the combined inhibition of YAP and FOSL-1 mRNA expression, using siRNA-lipoplexes, induces superior control over tumor growth in vitro and in vivo, compared to the individual treatments, and a reduction of the tumor stroma. The results offer a new therapeutic approach for pancreatic cancer treatment.Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer.

Single-cell transcriptome and accessible chromatin dynamics during endocrine pancreas development

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate β-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.

Insulin-degrading enzyme ablation in mouse pancreatic alpha cells triggers cell proliferation, hyperplasia and glucagon secretion dysregulation

Aims/hypothesisType 2 diabetes is characterised by hyperglucagonaemia and perturbed function of pancreatic glucagon-secreting alpha cells but the molecular mechanisms contributing to these phenotypes are poorly understood. Insulin-degrading enzyme (IDE) is present within all islet cells, mostly in alpha cells, in both mice and humans. Furthermore, IDE can degrade glucagon as well as insulin, suggesting that IDE may play an important role in alpha cell function in vivo.MethodsWe have generated and characterised a novel mouse model with alpha cell-specific deletion of Ide, the A-IDE-KO mouse line. Glucose metabolism and glucagon secretion in vivo was characterised; isolated islets were tested for glucagon and insulin secretion; alpha cell mass, alpha cell proliferation and α-synuclein levels were determined in pancreas sections by immunostaining.ResultsTargeted deletion of Ide exclusively in alpha cells triggers hyperglucagonaemia and alpha cell hyperplasia, resulting in elevated constitutive glucagon secretion. The hyperglucagonaemia is attributable in part to dysregulation of glucagon secretion, specifically an impaired ability of IDE-deficient alpha cells to suppress glucagon release in the presence of high glucose or insulin. IDE deficiency also leads to α-synuclein aggregation in alpha cells, which may contribute to impaired glucagon secretion via cytoskeletal dysfunction. We showed further that IDE deficiency triggers impairments in cilia formation, inducing alpha cell hyperplasia and possibly also contributing to dysregulated glucagon secretion and hyperglucagonaemia.Conclusions/interpretationWe propose that loss of IDE function in alpha cells contributes to hyperglucagonaemia in type 2 diabetes.Graphical abstract

ZD-2, a novel DPP4 inhibitor, protects islet β-cell and improves glycemic control in high-fat-diet-induced obese mice

AimsDipeptidyl peptidase-4 (DPP-4) inhibitors have been extensively used for the treatment of type 2 diabetes mellitus. Nevertheless, side effects like sore throat and diarrhea also occur in DPP-4 inhibitors treatment. The study aims to identify and develop novel DPP-4 inhibitors with better therapeutic profiles. Materials and methodsHere we synthesized a series of vildagliptin analogs, and among which, ZD-2 showed the moderate inhibition of DPP-4 activity compared with vildagliptin. High-fat-diet (HFD) mice were treated with ZD-2 (4.5 and 7.5 mg/kg) or vildagliptin (6 mg/kg) for 7 weeks following the examinations of metabolic index and pancreatic β-cell function. Mouse pancreatic cell line MIN6 was used to evaluate β-cell function, and intestinal enteroendocrine cell line STC-1 was used to evaluate the expression of gut hormones. Key findingsThe IC50 of ZD-2 was over 30-fold higher than vildagliptin. However, both ZD-2 and vildagliptin treatment showed comparable effects on improving glucose tolerance and reducing the steatosis of liver and fat mass in HFD mice. Moreover, ZD-2 exerted β-cell-protective actions by preserving islet β-cell mass and increasing the expression of functional β-cell-related genes. Additionally, ZD-2 also stimulated the expression of gut hormones in STC-1 cells. SignificanceZD-2 showed comparable anti-diabetic activities in HFD-fed mice although its lower potency on inhibition of DPP-4 compared with vildagliptin. Protection of β-cell function might contribute to its anti-diabetic effects.

GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca2+ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca2+ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.

Mutant Huntingtin Impairs Pancreatic β-cells by Recruiting IRS-2 and Disturbing the PI3K/AKT/FoxO1 Signaling Pathway in Huntington's Disease.

Patients with Huntington's disease (HD) have an increased incidence of diabetes. However, the molecular mechanisms of pancreatic β-cell dysfunction have not been entirely clarified. Revealing the pathogenesis of diabetes can provide a novel understanding of the onset and progression of HD, as well as potential clues for the development of new therapeutics. Here, we demonstrated that the mouse pancreatic insulinoma cell line NIT-1 expressing N-terminal mutant huntingtin (mHTT) containing 160 polyglutamine (160Q cells) displayed lower cell proliferative ability than the cells expressing N-terminal wild-type HTT containing 20 polyglutamine (20Q cells). In addition, 160Q cells were more prone to apoptosis and exhibited deficient glucose-stimulated insulin expression and secretion. Furthermore, insulin signaling molecule insulin receptor substrate 2 (IRS-2) expression decreased and was recruited into mHTT aggregates. Consequently, glucose stimulation failed to activate the downstream molecule phosphatidylinositol-3 kinase (PI3K) in 160Q cells, leading to reduced phosphorylation levels of serine-threonine protein kinase AKT and forkhead box protein O1 (FoxO1). These data indicate that activation of the glucose-stimulated PI3K/AKT/FoxO1 signaling pathway is significantly blocked in pancreatic β-cells in HD. Importantly, insulin treatment inhibited the aggregation of mHTT and significantly improved the activation of PI3K/AKT/FoxO1 signaling in 160Q cells. These results suggest that the inhibition of the PI3K/AKT/FoxO1 pathway might be due to the recruitment of IRS-2 into mHTT aggregates in HD β-cells, ultimately contributing to the impairment of pancreatic β-cells. In conclusion, our work provides new insight into the underlying mechanisms of the high incidence of diabetes and abnormal glucose homeostasis in HD.

Inhibition of lncRNA TCONS_00077866 Ameliorates the High Stearic Acid Diet-Induced Mouse Pancreatic β-Cell Inflammatory Response by Increasing miR-297b-5p to Downregulate SAA3 Expression.

Long-term consumption of a high-fat diet increases the circulating concentration of stearic acid (SA), which has a potent toxic effect on β-cells, but the underlying molecular mechanisms of this action have not been fully elucidated. Here, we evaluated the role of long noncoding (lnc)RNA TCONS_00077866 (lnc866) in SA-induced β-cell inflammation. lnc866 was selected for study because lncRNA high-throughput sequencing analysis demonstrated it to have the largest fold-difference in expression of five lncRNAs that were affected by SA treatment. Knockdown of lnc866 by virus-mediated shRNA expression in mice or by Smart Silencer in mouse pancreatic β-TC6 cells significantly inhibited the SA-induced reduction in insulin secretion and β-cell inflammation. According to lncRNA-miRNAs-mRNA coexpression network analysis and luciferase reporter assays, lnc866 directly bound to miR-297b-5p, thereby preventing it from reducing the expression of its target serum amyloid A3 (SAA3). Furthermore, overexpression of miR-297b-5p or inhibition of SAA3 also had marked protective effects against the deleterious effects of SA in β-TC6 cells and mouse islets. In conclusion, lnc866 silencing ameliorates SA-induced β-cell inflammation by targeting the miR-297b-5p/SAA3 axis. lnc866 inhibition may represent a new strategy to protect β-cells against the effects of SA during the development of type 2 diabetes.