mRNA Levels Of PERK Research Articles

Moutan cortex radicis extract alleviates lipid accumulation by modulating endoplasmic reticulum stress in high-fat diet-fed mice

To analyse the effects of Moutan cortex radicis extract (ME) on lipid metabolism disorder via regulation of lipid accumulation and endoplasmic reticulum stress (ERS). C57BL/6 mice were fed an HFD for 11 weeks and then an HFD plus 200 mg/kg ME for 2 weeks. We found that ME reduced hepatic CHOL levels and lipid droplet deposition, and down-regulated PPARγ, SCD1, GRP78 and C/EBP compared to the HFD group (P < 0.05). In the oleic acid-palmitic acid-induced AML12 cell lipid load model, ME addition led to significant reduction in the intracellular TG content, lipid deposition, and mRNA levels of PERK, IRE1, ATF4, CHOP, C/EBP-α, LXRα and ACC (P < 0.05). These findings indicate that ME might be a promising treatment strategy to reverse the lipid metabolism disorder induced by an HFD and that the potential mechanism is associated with a reduction in hepatic lipid accumulation and amelioration of ERS via inhibiting PERK/IRE1-CEBP signaling pathways.

Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark

Aims/hypothesisLevels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model.MethodsmiR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue.ResultsThe proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance.Conclusions/interpretationConcurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.Graphical abstract

The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading

This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved. L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully. The myoblasts were imposed tension for 0, 2, 6, 12 and 24 hours respectively by multi-channel cell stress loading system. The force value was 15% cell deformation and the frequency was 10 cycles/min. Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds, and the group without tension was used as the control group. The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate; the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot. PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch. SPSS 17.0 software package was used to analyze the data statistically. DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast. Apoptosis rate showed a trend of rising gradually over time, peaked at 24 h. After dealt with the inhibitor of PERK, the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control. The results of real- time PCR showed that the mRNA of CHOP was increased with the extension loading time, while the mRNA of PERK showed no difference compared with the control. Western blot results showed that the protein level of p-PERK was increased with the extension of loading time, while the expression of PERK showed no difference compared with the control group. When PERK inhibitor added, the mRNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control. PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch, and the possible mechanism may be closely related to the phosphorylation of PERK.

Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma

Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1α and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1α and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1α transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1α and PERK, in PEL cells. However the IRE1α and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1α and PERK in PEL cells, transcriptional suppression of IRE1α by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress.