Differential effects of three echovirus strains on cell lysis and insulin secretion in beta cell derived lines

Sarmiento, Luis; Medina, Anya; Aziz, Kosrat; Anagandula, Mahesh; Cabrera-Rode, Eduardo; Fex, Malin; Frisk, Gun; Cilio, Corrado

doi:10.1002/jmv.24438

Cited by 11 publications

(12 citation statements)

References 33 publications

(48 reference statements)

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“…Previous studies have shown that strains of E16, isolated from patients with meningitis, resulted in the development of diabetes-related islet autoantibodies [18,19,25]. In addition, E16 is able to replicate in explanted human islets and clonal beta cell lines, thus concluding that E16 can target pancreatic endocrine cells [26,27].…”

Section: Discussionmentioning

confidence: 98%

Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

et al. 2020

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Aim/hypothesis Human enteroviral infections are suggested to be associated with type 1 diabetes. However, the mechanism by which enteroviruses can trigger disease remains unknown. The present study aims to investigate the impact of enterovirus on autophagy, a cellular process that regulates beta cell homeostasis, using the clonal beta cell line INS(832/13) and human islet cells as in vitro models. Methods INS(832/13) cells and human islet cells were infected with a strain of echovirus 16 (E16), originally isolated from the stool of a child who developed type 1 diabetes-associated autoantibodies. Virus production and release was determined by 50% cell culture infectious dose (CCID50) assay and FACS analysis. The occurrence of autophagy, autophagosomes, lysosomes and autolysosomes was detected by western blot, baculoviral-mediated expression of microtubule-associated protein light chain 3 (LC3)II-GFP and LysoTracker Red, and quantified by Cellomics ArrayScan. Autophagy was also monitored with a Cyto-ID detection kit. Nutrient deprivation (low glucose [2.8 mmol/l]), amino acid starvation (Earle’s Balanced Salt Solution [EBSS]) and autophagy-modifying agents (rapamycin and chloroquine) were used in control experiments. Insulin secretion and the expression of autophagy-related (Atg) genes and genes involved in autophagosome–lysosome fusion were determined. Results E16-infected INS(832/13) cells displayed an accumulation of autophagosomes, compared with non-treated (NT) cells (grown in complete RPMI1640 containing 11.1 mmol/l glucose) (32.1 ± 1.7 vs 21.0 ± 1.2 μm2/cell; p = 0.05). This was accompanied by increased LC3II ratio both in E16-infected cells grown in low glucose (LG) (2.8 mmol/l) (0.42 ± 0.03 vs 0.11 ± 0.04 (arbitrary units [a.u.]); p < 0.0001) and grown in media containing 11.1 mmol/l glucose (0.37 ± 0.016 vs 0.05 ± 0.02 (a.u.); p < 0.0001). Additionally, p62 accumulated in cells after E16 infection when grown in LG (1.23 ± 0.31 vs 0.36 ± 0.12 (a.u.); p = 0.012) and grown in media containing 11.1 mmol/l glucose (1.79 ± 0.39 vs 0.66 ± 0.15 (a.u.); p = 0.0078). mRNA levels of genes involved in autophagosome formation and autophagosome–lysosome fusion remained unchanged in E16-infected cells, except Atg7, which was significantly increased when autophagy was induced by E16 infection, in combination with LG (1.48 ± 0.08-fold; p = 0.02) and at 11.1 mmol/l glucose (1.26 ± 0.2-fold; p = 0.001), compared with NT controls. Moreover, autophagosomes accumulated in E16-infected cells to the same extent as when cells were treated with the lysosomal inhibitor, chloroquine, clearly indicating that autophagosome turnover was blocked. Upon infection, there was an increased viral titre in the cell culture supernatant and a marked reduction in glucose-stimulated insulin secretion (112.9 ± 24.4 vs 209.8 ± 24.4 ng [mg protein]–1 h–1; p = 0.006), compared with uninfected controls, but cellular viability remained unaffected. Importantly, and in agreement with the observations for INS(832/13) cells, E16 infection impaired autophagic flux in primary human islet cells (46.5 ± 1.6 vs 34.4 ± 2.1 μm2/cell; p = 0.01). Conclusions/interpretation Enteroviruses disrupt beta cell autophagy by impairing the later stages of the autophagic pathway, without influencing expression of key genes involved in core autophagy machinery. This results in increased viral replication, non-lytic viral spread and accumulation of autophagic structures, all of which may contribute to beta cell demise and type 1 diabetes.

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Section: Discussionmentioning

confidence: 98%

Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

et al. 2020

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“…Human enteroviruses replication in islets and ductal—but not in acinar—cells has been demonstrated in earlier reports [ 3 , 18 , 19 , 32 , 33 ], suggesting that human exocrine pancreas may not represent a target of HEV. Accordingly, we previously showed by virus titration studies, immunoreactivity of HEV VP1 protein and mRNA expression of innate immunity genes that E16 and E30 can productively infect both primary human islets and β cell-derived lines [ 21 , 22 ]. The present study confirms this observation by revealing an increase in viral titer when islets were infected with E16 and E30.…”

Section: Discussionmentioning

confidence: 99%

“…However, most of the aforementioned studies have focused mainly on CVB, and there has been no attempt to assess the possible tropism of echovirus towards exocrine cells. Although we previously demonstrated that strains of E16 and E30 with a proven association with islet autoimmunity were able to replicate in explanted human islets and β cell-derived lines [ 20 , 21 , 22 ], their effect on human exocrine cells has never been tested. Interestingly, maternal infection with E6, a closely related echovirus strain, was associated with the induction of coexisting neonatal type 1 diabetes and exocrine pancreatic insufficiency in a Finnish report [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

Echovirus 6 Infects Human Exocrine and Endocrine Pancreatic Cells and Induces Pro-Inflammatory Innate Immune Response

Sarmiento

Frisk

Anagandula

et al. 2017

Viruses

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Human enteroviruses (HEV), especially coxsackievirus serotype B (CVB) and echovirus (E), have been associated with diseases of both the exocrine and endocrine pancreas, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. This study aimed to investigate the capability of echovirus strains to infect human exocrine and endocrine pancreatic cells. Infection of explanted human islets and exocrine cells with seven field strains of E6 caused cytopathic effect, virus titer increase and production of HEV protein VP1 in both cell types. Virus particles were found in islets and acinar cells infected with E6. No cytopathic effect or infectious progeny production was observed in exocrine cells exposed to the beta cell-tropic strains of E16 and E30. Endocrine cells responded to E6, E16 and E30 by upregulating the transcription of interferon-induced with helicase C domain 1 (IF1H1), 2′-5′-oligoadenylate synthetase 1 (OAS1), interferon-β (IFN-β), chemokine (C–X–C motif) ligand 10 (CXCL10) and chemokine (C–C motif) ligand 5 (CCL5). Echovirus 6, but not E16 or E30, led to increased transcription of these genes in exocrine cells. These data demonstrate for the first time that human exocrine cells represent a target for E6 infection and suggest that certain HEV serotypes can replicate in human pancreatic exocrine cells, while the pancreatic endocrine cells are permissive to a wider range of HEV.

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“…After the medium was discarded, cells were preincubated with Krebs‐Ringer bicarbonate HEPES (KRBH) buffer (140 mmol/L NaCl, 3.6 mmol/L KCl, 0.5 mmol/L NaH 2 PO 4 , 0.5 mmol/L MgSO 4 , 1.5 mmol/L CaCl 2 , 2 mmol/L NaHCO 3 , 10 mmol/L HEPES, and 0.1% bovine serum albumin) for 2 hours at 37°C. According to a previous report, morin was added to the KRBH buffer containing various concentrations of glucose. The plate was then incubated for 2 hours at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…According to a previous report, 33 morin was added to the KRBH buffer containing various concentrations of glucose. The plate was then incubated for 2 hours at 37°C.…”

Section: Glucose-stimulated Insulin Secretion (Gsis)mentioning

confidence: 99%

Investigation of morin‐induced insulin secretion in cultured pancreatic cells

Lin

Hsu

Lin

et al. 2017

Clin Exp Pharma Physio

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Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetes. Insulin secretion has been demonstrated to increase following the administration of morin. The present study is designed to investigate the potential mechanism(s) of morin-induced insulin secretion in the MIN6 cell line. First, we identified that morin induced a dose-dependent increase in insulin secretion and intracellular calcium content in MIN6 cells. Morin potentiated glucose-stimulated insulin secretion (GSIS). Additionally, we used siRNA for the ablation of imidazoline receptor protein (NISCH) expression in MIN6 cells. Interestingly, the effects of increased insulin secretion by morin and canavanine were markedly reduced in Si-NISCH cells. Moreover, we used KU14R to block imidazoline I receptor (I-3R) that is known to enhance insulin release from the pancreatic β-cells. Without influence on the basal insulin secretion, KU14R dose-dependently inhibited the increased insulin secretion induced by morin or efaroxan in MIN6 cells. Additionally, effects of increased insulin secretion by morin or efaroxan were reduced by diazoxide at the dose sufficient to open K channels and attenuated by nifedipine at the dose used to inhibit L-type calcium channels. Otherwise, phospholipase C (PLC) is introduced to couple with imidazoline receptor (I-R). The PLC inhibitor dose-dependently inhibited the effects of morin in MIN6 cells. Similar blockade was also observed in protein kinase C (PKC) inhibitor-treated cells. Taken together, we found that morin increases insulin secretion via the activation of I-R in pancreatic cells. Therefore, morin would be useful to develop in the research and treatment of diabetic disorders.

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Differential effects of three echovirus strains on cell lysis and insulin secretion in beta cell derived lines

Cited by 11 publications

References 33 publications

Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

Echovirus 6 Infects Human Exocrine and Endocrine Pancreatic Cells and Induces Pro-Inflammatory Innate Immune Response

Investigation of morin‐induced insulin secretion in cultured pancreatic cells

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