Amino acid restriction alters survival mechanisms in pancreatic beta cells: possible role of the PI3K/Akt pathway.
Ontology highlight
ABSTRACT: Background and aims
Malnutrition in the early stages of life may lead to changes in the glycemic metabolism during adulthood, such as pancreatic beta cells dysfunction and failure. Therefore, this study aimed to evaluate the effects of an in vitro amino acid restriction model on the function and viability of pancreatic beta cells.Methods
Insulin-producing cells (INS-1E) were maintained in control or amino acid restricted culture medium containing 1 × or 0.25 × of amino acids, respectively, for 48 h.Results
Amino acid restricted group showed lower insulin secretion and insulin gene expression, reduced mitochondrial oxygen consumption rate and reactive oxygen species production. Besides, amino acid restricted group also showed higher levels of endoplasmic reticulum stress and apoptosis markers and enhanced Akt phosphorylation. However, even with higher levels of apoptosis markers, amino acid restricted group did not show higher levels of cell death unless the PI3K/Akt pathway was inhibited.Conclusion
Amino acid restricted beta cell viability seems to be dependent on the PI3K/Akt pathway.
SUBMITTER: Alves BL
PROVIDER: S-EPMC8081284 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:ObjectiveMicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains challenging. In this study, we aimed to identify miRNAs and their downstream targets involved in the regeneration of islet beta cells following partial pancreatectomy in mice.MethodsRNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of the selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA, protein expression levels, and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice.ResultsPartial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed that 14 miRNAs, including miR-132 and -141, were significantly upregulated in the LCM islets of the partially pancreatectomized mice compared to the LCM islets of the control mice. In the same comparison, miR-760 was the only downregulated miRNA. The changed expression of these miRNAs in the islets of the partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. The opposite was observed in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR, and immunoblotting of the latter cells demonstrated their downregulated expression of Pten with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb and inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in the LCM islets of pancreatectomized mice compared to the sham-operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-βH1 cells. The regeneration of beta cells following partial pancreatectomy was lower in the miR-132/212-/- mice than the control littermates.ConclusionsThis study provides compelling evidence about the critical role of miR-132 for the regeneration of mouse islet beta cells through the downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.
| S-EPMC6928290 | biostudies-literature
Project description:Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than 6 months. Oncogenic alterations in ATC include aberrant phosphoinositide 3 kinase (PI3K) signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and protein kinase B (Akt) amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRβ) is strongly associated with ATC. TRβ is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85α. However, the role of TRβ in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRβ indeed suppresses PI3K signaling in ATC cell lines through unreported genomic mechanisms, including a decrease in RTK expression and an increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRβ in ATC cell lines enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRβ but shed light on the implication of TRβ status and activation on inhibitor efficacy in ATC tumors.
| S-EPMC8271203 | biostudies-literature
Project description:Background:Transcription factor 7-like 2 (TCF7L2), which previously known as TCF-4, is a major form of transcription factor involved in the downstream WNT signaling and exhibits the strongest association to diabetes susceptibility. Although we still do not know mechanistically how TCF7L2 exerts its physiological functions on pancreatic endocrine cells, it had been suggested that TCF7L2 may directly affect ?-cell function by regulating the activation of PI3K/AKT signaling pathway. Methods:MIN6 cells were transfected with TCF7L2 knockdown virus or lenti-TCF7L2 virus for 48 h to evaluate the contribution of TCF7L2 to the PI3K/AKT signaling pathway and pancreatic ?-cell function. This was confirmed by measuring the expression of PI3K p85 and p-Akt by western blotting and insulin secretion by enzyme-linked immunosorbent assay (ELISA), respectively. Chromatin immunoprecipitation (ChIP) and polymerase chain reaction (PCR) experiments were performed to explore the genomic distribution of TCF7L2-binding sites in the promoter of PIK3R1, the affinity between which was analyzed by the luciferase reporter assay. Results:In the present study, we strikingly identified that TCF7L2 could profoundly inhibit the expression of PIK3R1 gene and its encoding protein PI3K p85, which then could lead to the activation of PI3K/AKT signaling and stimulate insulin secretion in pancreatic ?-cells. However, the integrity and stability of evolutionarily conserved TCF7L2-binding motif plays a very crucial role in the binding events between transcription factor TCF7L2 and its candidate target genes. We also found that the affinity of TCF7L2 to the promoter region of PIK3R1 alters upon the specific binding sites, which further provides statistical validation to the necessity of TCF7L2-binding motif. Conclusions:This study demonstrated that TCF7L2 is closely bound to the specific binding regions of PIK3R1 promoter and prominently controls the transcription of its encoding protein p85, which further affects the activation of PI3K/AKT signaling pathway and insulin secretion.
| S-EPMC6612183 | biostudies-literature
Project description:BackgroundThe survival of pancreatic cancer patients remains poor. However, the underlying molecular mechanism and new therapeutic target of pancreatic cancer are still needed to be found. Many studies have shown that the IGF2 mRNA-binding protein 2 (IGF2BP2) plays oncogenic roles in cancers. However, the clinical significance, role and molecular mechanisms of IGF2BP2 in pancreatic cancer remain unclear.MethodsThe expression of IGF2BP2 and miR-141 was detected in pancreatic cancer, and clinical significances were analyzed by statistical analysis. The function of IGF2BP2 and miR-141 was determined in vitro and in vivo, and the underlying mechanism was investigated. The gene copy number variation (CNV) of IGF2BP2 was analyzed based on The Cancer Genome Atlas (TCGA) dataset. microRNAs (miRNAs) regulating IGF2BP2 were predicted by online tools and confirmed by experiments.ResultsIGF2BP2 is overexpressed in pancreatic cancer tissues compared with control tissues. Upregulation of IGF2BP2 predicts shorter overall survival (OS) in pancreatic cancer patients by statistical analysis. IGF2BP2 overexpression is partially due to genomic amplification. Bioinformatics analyses and validation experiments showed that IGF2BP2 is a direct target of miR-141. A negative correlation between IGF2BP2 mRNA expression and the expression of miR-141 was observed in pancreatic cancer tissues and more importantly, reexpression of miR-141 rescued the oncogenic role of IGF2BP2. Moreover, upregulating IGF2BP2 expression promotes pancreatic cancer cell growth by activating the PI3K/Akt signaling pathway in vitro and in vivo.ConclusionsWe comprehensively reveal the oncogenic role of IGF2BP2 in pancreatic cancer carcinogenesis and confirm that genomic amplification and the silencing of miR-141 contribute to its activation. Our findings highlight that IGF2BP2 may be a promising molecular target for the treatment of pancreatic cancer.
| S-EPMC6921559 | biostudies-literature
Project description:In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.
| S-EPMC3471824 | biostudies-literature
Project description:BackgroundElevated uric acid (UA) has been found to damage pancreatic β-cell, promote oxidative stress, and cause insulin resistance in type 2 diabetes (T2D). Astragaloside IV (AS-IV), a major active monomer extracted from Astragalus membranaceus (Fisch.) Bunge. which belongs to TRIB. Galegeae (Br.) Torrey et Gray, Papilionaceae, exhibits various activities in a pathophysiological environment and has been widely employed to treat diseases. However, the effects of AS-IV on UA-induced pancreatic β-cell damage need to be investigated and the associating mechanism needs to be elucidated. This study was designed to determine the protective effects and underlying mechanism of AS-IV on UA-induced pancreatic β-cell dysfunction in T2D.MethodsUA-treated Min6 cells were exposed to AS-IV or wortmannin. Thereafter, the 3-(45)-dimethylthiahiazo(-z-y1)-35-di-phenytetrazoliumromide (MTT) assay and flow cytometry were employed to determine the effect of AS-IV on cell proliferation and apoptosis, respectively. Insulin secretion was evaluated using the glucose-stimulated insulin secretion (GSIS) assay. Finally, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to determine the effect of AS-IV on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in UA-treated cells.ResultsAS-IV had no cytotoxic effects on Min6 cells. UA significantly suppressed Min6 cell growth, promoted cell apoptosis, and enhanced caspase-3 activity; however, AS-IV abolished these effects in a dose-dependent manner. Further, decreased insulin secretion was found in UA-treated Min6 cells compared to control cells, and the production of insulin was enhanced by AS-IV in a dose-dependent manner. AS-IV significantly increased phosphorylated (p)-AKT expression and the ratio of p-AKT/AKT in Min6 cells exposed to UA. No evident change in AKT mRNA level was found in the different groups. However, the effects of AS-IV on UA-stimulated Min6 cells were reversed by 100 nM wortmannin.ConclusionCollectively, our data suggest that AS-IV protected pancreatic β-cells from UA-treated dysfunction by activating the PI3K/AKT pathway. Such findings suggest that AS-IV may be an efficient natural agent against T2D.
| S-EPMC8763508 | biostudies-literature
Project description:Recent studies have linked branched-chain amino acid (BCAA) with numerous metabolic diseases. However, the molecular basis of BCAA's roles in metabolic regulation remains to be established. KLF15 (Krüppel-like factor 15) is a transcription factor and master regulator of glycemic, lipid, and amino acids metabolism. In the present study, we found high concentrations of BCAA suppressed KLF15 expression while BCAA starvation induced KLF15 expression, suggesting KLF15 expression is negatively controlled by BCAA.Interestingly, BCAA starvation induced PI3K-AKT signaling. KLF15 induction by BCAA starvation was blocked by PI3K and AKT inhibitors, indicating the activation of PI3K-AKT signaling pathway mediated the KLF15 induction. BCAA regulated KLF15 expression at transcriptional level but not post-transcriptional level. However, BCAA starvation failed to increase the KLF15-promoter-driven luciferase expression, suggesting KLF15 promoter activity was not directly controlled by BCAA. Finally, fasting reduced BCAA abundance in mice and KLF15 expression was dramatically induced in muscle and white adipose tissue, but not in liver. Together, these data demonstrated BCAA negatively regulated KLF15 expression, suggesting a novel molecular mechanism underlying BCAA's multiple functions in metabolic regulation.
| S-EPMC5661165 | biostudies-literature
Project description:BackgroundCarboxypeptidase A4 (CPA4), as a novel tumor biomarker, is prevalently observed in various cancers. However, the potential role of CPA4 in pancreatic cancer (PC), to our knowledge, has not been fully clarified.Materials and methodsWe systematically explored the detailed function of CPA4 in epithelial to mesenchymal transition (EMT) stimulated PC in human clinical samples and in vitro.ResultsCPA4 was overexpressed in clinical PC samples that was positively related with tumor size (P=0.026), T stage (P=0.011), lymph-node metastasis (P=0.026) and a worse prognosis for PC patients (P=0.001). Interestingly, CPA4 was inversely correlated with E-cadherin (r=-0.372, P=0.003) in clinical samples and PC cell lines which cooperatively contributed to a worse prognosis (P=0.005) for PC patients. CPA4 overexpression enhanced EMT in AsPC-1 and Capan-2 cells, which promoted EMT-like cellular morphology and cell invasion and migration. Meanwhile, CPA4 overexpression activated EMT and PI3K-AKT-mTOR signaling, following with the downregulation of E-cadherin and β-catenin, and the upregulation of N-cadherin, vimentin, p-PI3K (Tyr458), p-AKT (Ser473) and p-mTOR (Ser2448). However, PI3K inhibitor LY294002 reversed CPA4 overexpression-stimulated EMT in vitro. Moreover, CPA4 was co-immunoprecipitated with AKT in two PC cells with CPA4 high expression. Conversely, CPA4 silencing inhibited EMT in PANC-1 cells. CPA4 overexpression or silencing promoted or inhibited cell proliferation and drug resistance in Capan-2 and PANC-1 cells via regulating Bcl2/Bax and cleaved-caspase3 signaling. However, LY294002 reversed CPA4 overexpression-stimulated cell proliferation and drug resistance in vitro in Bcl2/Bax and caspase3-dependent apoptosis.ConclusionCPA4 overexpression contributes to aggressive clinical stage of PC patients and promotes EMT in vitro via activation of PI3K-AKT-mTOR signaling.
| S-EPMC7457871 | biostudies-literature
Project description:Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans, CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore, our data identify promising pathways for therapeutic targets to combat age-related diseases and promote health in humans.
| S-EPMC3714316 | biostudies-literature
Project description:The present work describes the anti-invasive effect of conjugate BC06, a novel conjugate of EPA, (2E,4E)-4-(benzo[d][1,3]dioxol-5-ylmethylene) hex-2-enoic acid with β,β-disubstituted-β-amino acid, β(3,3)-Pip-OH (2-(4-aminopiperidin-4-yl)acetic acid), in human pancreatic carcinoma. The conjugate BC06 inhibited invasion and migration of PANC-1 cells in wound healing, matrigel invasion, and gelatin degradation assays. Apart from suppressing PI3K/Akt/NF-kB signaling, which is involved in the up-regulation of matrix metalloproteinases, our study also demonstrated that dose-dependent treatment of BC06 results in the upregulation of TIMP-1 and E-cadherin expression. Further, BC06 was found to be inhibiting the metastatic ability of PANC-1 cells by reducing MMP-2 and MMP-9 expression. These findings suggest that EPA conjugate with β(3,3)-Pip-OH, BC06, may be used as an anti-invasive agent against human pancreatic carcinoma.
| S-EPMC4601056 | biostudies-literature