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Roles of oxidative stress, apoptosis, and inflammation in metal-induced dysfunction of beta pancreatic cells isolated from CD1 mice.

ABSTRACT: The diabetogenic effects of metals including lead (Pb), mercury (Hg), cadmium (Cd), and molybdenum (Mo) have been reported with poorly identified underlying mechanisms. The current study assessed the effect of metals on the roles of oxidative stress, apoptosis, and inflammation in beta pancreatic cells isolated from CD-1 mice, via different biochemical assays. Data showed that the tested metals were cytotoxic to the isolated cells with impaired glucose stimulated insulin secretion (GSIS). This was associated with increased reactive oxygen species (ROS) production, lipid peroxidation, antioxidant enzymes activities, active proapoptotic caspase-3 (cas-3), inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels in the intoxicated cells. Furthermore, antioxidant-reduced glutathione (GSH-R), cas-3 inhibitor z-VAD-FMK, IL-6 inhibitor bazedoxifene (BZ), and TNF-α inhibitor etanercept (ET) were found to significantly decrease metal-induced cytotoxicity with improved GSIS in metals' intoxicated cells. In conclusion, oxidative stress, apoptosis, and inflammation can play roles in metals-induced diabetogenic effect.

SUBMITTER: Al Doghaither H

PROVIDER: S-EPMC7785459 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Roles of oxidative stress, apoptosis, and inflammation in metal-induced dysfunction of beta pancreatic cells isolated from CD1 mice.

Al Doghaither Huda H Elmorsy Ekramy E Al-Ghafari Ayat A Ghulam Jihan J

Saudi journal of biological sciences 20201104 1

The diabetogenic effects of metals including lead (Pb), mercury (Hg), cadmium (Cd), and molybdenum (Mo) have been reported with poorly identified underlying mechanisms. The current study assessed the effect of metals on the roles of oxidative stress, apoptosis, and inflammation in beta pancreatic cells isolated from CD-1 mice, via different biochemical assays. Data showed that the tested metals were cytotoxic to the isolated cells with impaired glucose stimulated insulin secretion (GSIS). This w ...[more]

PMID: 33424352

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Project description:Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells, and it primarily functions to regulate cell signaling, glycolipid metabolism and apoptosis. The catalytic subunit of PP2A (PP2Ac) plays an important role in the functions of the protein. However, there are few reports on the regulatory role of PP2Ac in pancreatic β-cells under lipotoxic conditions. In the present study, mouse insulinoma 6 (MIN6) pancreatic cells were transfected with short hairpin RNAs to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish a lipotoxicity model. Serine/threonine phosphatase assay system, Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay and western blotting were used to measure PP2A activity, cell viability, apoptosis, oxidative stress and insulin secretion in the cells. In addition, a mouse model of lipotoxicity was established with a high-fat diet (HFD) and the knockdown of PP2Ac using adeno-associated viruses to interfere with PP2Ac expression in the pancreatic tissues. The activity of PP2A in the mouse pancreatic tissue and the serum insulin level were measured. Furthermore, the proliferation of mouse pancreatic β-cells was assessed using pancreatic tissue immunofluorescence. PP2Ac knockdown inhibited lipotoxicity-induced PP2A hyperactivation, increased the resistance of pancreatic β-cells to lipotoxicity and attenuated PA-induced apoptosis in MIN6 cells. It also protected the endoplasmic reticulum and mitochondria, and ameliorated insulin secretion. The results of mRNA sequencing and western blotting analysis suggested that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated via the MAPK pathway. Moreover, the results of the animal experiments suggested that specific knockdown of pancreatic PP2Ac effectively attenuated HFD-induced insulin resistance and reduced the compensatory proliferation of pancreatic β-cells in mice. In summary, the present study revealed the effects of interfering with PP2Ac gene expression on pancreatic β-cells in vivo and in vitro and the underlying mechanisms, which may provide insights for the treatment of type 2 diabetes mellitus in the clinic.

Project description:BackgroundInflammation, oxidative stress, and apoptosis contribute to the evolution of doxorubicin (DOX)-induced cardiotoxicity. Osteocrin (OSTN) is a novel secretory peptide mainly derived from the bone and skeletal muscle, and plays critical roles in regulating bone growth and physical endurance. Inspiringly, OSTN was also reported to be abundant in the myocardium that functioned as a therapeutic agent against cardiac rupture and congestive heart failure in mice after myocardial infarction. Herein, we investigated the role and potential mechanism of OSTN in DOX-induced cardiotoxicity.MethodsCardiac-restrict OSTN overexpression was performed by the intravenous injection of a cardiotropic AAV9 vector, and subsequently the mice received 15 mg/kg DOX injection (i.p., once) to induce acute cardiac injury. Besides, H9C2 cell lines were used to assess the possible role of OSTN in vitro by incubating with recombinant human OSTN or small interfering RNA against Ostn (siOstn). To clarify the involvement of protein kinase G (PKG), KT5823 and siPkg were used in vivo and in vitro. Mice were also administrated intraperitoneally with 5 mg/kg DOX weekly for consecutive 3 weeks at a cumulative dose of 15 mg/kg to mimic the cardiotoxic effects upon chronic DOX exposure.ResultsOSTN treatment notably attenuated, whereas OSTN silence exacerbated inflammation, oxidative stress, and cardiomyocyte apoptosis in DOX-treated H9C2 cells. Besides, cardiac-restrict OSTN-overexpressed mice showed an alleviated cardiac injury and malfunction upon DOX injection. Mechanistically, we found that OSTN activated PKG, while PKG inhibition abrogated the beneficial effect of OSTN in vivo and in vitro. As expected, OSTN overexpression also improved cardiac function and survival rate in mice after chronic DOX treatment.ConclusionsOSTN protects against DOX-elicited inflammation, oxidative stress, apoptosis, and cardiac dysfunction via activating PKG, and cardiac gene therapy with OSTN provides a novel therapeutic strategy against DOX-induced cardiotoxicity.

Project description:Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells and primarily functions to regulate cell signaling, glycolipid metabolism, and apoptosis. Its PP2A catalytic subunit (PP2Ac) plays an important role in its function. Nonetheless, at present, there are only a few reports on the regulatory role of PP2Ac in pancreatic β-cells under lipotoxicity. Mouse pancreatic insulinoma (MIN6) cells were transfected by lentiviruses to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish the lipotoxicity model. Serine/Threonine Phosphatase Assay System kit, Cell Counting Kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB) and other techniques were used to measure PP2A activity, cell viability, apoptosis, oxidative stress, and insulin secretion. An animal model of lipotoxicity with knockdown of PP2Ac was established by a high-fat diet (HFD) after using adeno-associated viruses (AAV) to interfere with PP2Ac expression in mouse pancreatic tissues. Serine/Threonine Phosphatase Assay System kit, ELISA, pancreatic tissue immunofluorescence and other techniques were used to measure PP2A activity in pancreatic tissue, serum insulin level and the proliferation of mouse pancreatic β-cells. We found that PP2Ac knockdown inhibited lipotoxicity-induced PP2A hyperactivation, increased the resistance of pancreatic β-cells to lipotoxicity, decreased PA-induced apoptosis in MIN6 cells, protected the function of both the endoplasmic reticulum (ER) and mitochondria, and improved insulin secretion. By mRNA sequencing and Western blotting analysis, we hypothesized that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated by the MAPK pathway. Moreover, the results obtained from animal experiments suggest that the specific knockdown of pancreatic PP2Ac could effectively attenuate HFD-induced insulin resistance and reduce the compensatory proliferation of pancreatic β-cells in mice. The present study revealed the effects and mechanisms of interfering with PP2Ac gene expression on pancreatic β-cells in vivo and in vitro, which might provide insights for the treatment of type 2 diabetes in the clinic.

Dataset Information

Roles of oxidative stress, apoptosis, and inflammation in metal-induced dysfunction of beta pancreatic cells isolated from CD1 mice.

Publications

Roles of oxidative stress, apoptosis, and inflammation in metal-induced dysfunction of beta pancreatic cells isolated from CD1 mice.

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