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Metabolome response to glucose in the ?-cell line INS-1 832/13.

ABSTRACT: Glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 ?-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

SUBMITTER: Lorenz MA

PROVIDER: S-EPMC3624472 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Metabolome response to glucose in the β-cell line INS-1 832/13.

Lorenz Matthew A MA El Azzouny Mahmoud A MA Kennedy Robert T RT Burant Charles F CF

The Journal of biological chemistry 20130220 15

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to ...[more]

PMID: 23426361

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Project description:Aim/hypothesisHuman 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.MethodsINS(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.ResultsE16-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/interpretationEnteroviruses 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. Graphical abstract.

Project description:Inorganic arsenic (iAs) is an environmental diabetogen, but mechanisms underlying its diabetogenic effects are poorly understood. Exposures to arsenite (iAsIII) and its methylated metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), have been shown to inhibit glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and isolated pancreatic islets. GSIS is regulated by complex mechanisms. Increase in ATP production through metabolism of glucose and other substrates is the ultimate trigger for GSIS in β-cells. In the present study, we used metabolomics to identify metabolites and pathways perturbed in cultured INS-1 832/13 rat insulinoma cells and isolated murine pancreatic islets by exposures to iAsIII, MAsIII and DMAsIII. We found that the exposures perturbed multiple metabolites, which were enriched primarily in the pathways of amino acid, carbohydrate, phospholipid and carnitine metabolism. However, the effects of arsenicals in INS-1 832/13 cells differed from those in the islets and were exposure specific with very few overlaps between the three arsenicals. In INS-1 832/13 cells, all three arsenicals decreased succinate, a metabolite of Krebs cycle, which provides substrates for ATP synthesis in mitochondria. Acetylcarnitine was decreased consistently by exposures to arsenicals in both the cells and the islets. Acetylcarnitine is usually found in equilibrium with acetyl-CoA, which is the central metabolite in the catabolism of macronutrients and the key substrate for Krebs cycle. It is also thought to play an antioxidant function in mitochondria. Thus, while each of the three trivalent arsenicals perturbed specific metabolic pathways, which may or may not be associated with GSIS, all three arsenicals appeared to impair mechanisms that support ATP production or antioxidant defense in mitochondria. These results suggest that impaired ATP production and/or mitochondrial dysfunction caused by oxidative stress may be the mechanisms underlying the inhibition of GSIS in β-cells exposed to trivalent arsenicals.

Dataset Information

Metabolome response to glucose in the ?-cell line INS-1 832/13.

Publications

Metabolome response to glucose in the β-cell line INS-1 832/13.

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