BPS-induced ovarian dysfunction: Protective actions of melatonin via modulation of SIRT-1/Nrf2/NFĸB and IR/PI3K/pAkt/GLUT-4 expressions in adult golden hamster.

Ever-increasing occurrence of plastic-manufacturing industries leads to environmental pollution that has been associated with declined human health and increased incidence of compromised reproductive health. Female subfertility/infertility is a complex phenomenon and environmental toxicants as well as lifestyle factors have a crucial role to play. Bisphenol S (BPS) was believed to be a "safer" replacement of bisphenol A (BPA) but recent data documented its neurotoxic, hepatotoxic, nephrotoxic, and reprotoxic attributes. Hence based on the scarcity of reports, we investigated molecular insights into BPS-induced ovarian dysfunction and protective actions of melatonin against it in adult golden hamsters, Mesocricetus auratus. Hamsters were administered with melatonin (3 mg/kg BW i.p. alternate days) and BPS (150 mg/kg BW orally every day) for 28 days. BPS treatment disrupted hypothalamo-pituitary-ovarian (HPO) axis as evident by reduced gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), ovarian steroids such as estradiol (E2) and progesterone (P4), thyroid hormones namely triiodothyronine (T3) and thyroxine (T4) and melatonin levels along with their respective receptors (ERα, TRα, and MT-1) thereby reducing ovarian folliculogenesis. BPS exposure also led to ovarian oxidative stress/inflammation by increasing reactive oxygen species and metabolic disturbances. However, melatonin supplementation to BPS restored ovarian folliculogenesis/steroidogenesis as indicated by increased number of growing follicles/corpora lutea and E2/P4 levels. Further, melatonin also stimulated key redox/survival markers such as silent information regulator of transcript-1 (SIRT-1), forkhead box O-1 (FOXO-1), nuclear factor E2-related factor-2 (Nrf2), and phosphoinositide 3-kinase/protein kinase B (PI3K/pAkt) expressions along with enhanced ovarian antioxidant capacity. Moreover, melatonin treatment reduced inflammatory load including ovarian nuclear factor kappa-B (NFĸB), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expressions, serum tumor necrosis factor α (TNFα), C-reactive protein (CRP) and nitrite-nitrate levels as well as upregulated ovarian insulin receptor (IR), glucose uptake transporter-4 (GLUT-4), connexin-43, and proliferating cell nuclear antigen (PCNA) expressions in ovary thereby ameliorating inflammatory and metabolic alterations due to BPS. In conclusion, we found severe deleterious impact of BPS on ovary while melatonin treatment protected ovarian physiology from these detrimental changes suggesting it to be a potential preemptive candidate against environmental toxicant-compromised female reproductive health.

Reversal of cisplatin resistance by neferine/isoliensinine and their combinatorial regimens with cisplatin-induced apoptosis in cisplatin-resistant colon cancer stem cells (CSCs).

Cisplatin chemotherapy to the colorectal cancer cells (CRCs) is accompanied by dose-limiting adverse effects along with the acquisition of drug resistance implicating low therapeutic outcomes. The present study is aimed to evaluate the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimen of neferine/isoliensinine with cisplatin against CSCs (cisplatin resistant colon stem cells). CSCs were developed using pulse exposure of cisplatin to parental HCT-15 cells. Neferine/isoliensinine or combinatorial regimens of Neferine/isoliensinine and cisplatin exhibited a stronger cytotoxic activity against CSCs compared to control. IC50 doses were found to be 6.5 µM for neferine, 12.5 µM for isoliensinine, and 120 µM for cisplatin respectively. Furthermore, the combinatorial regimen of a low dose of cisplatin (40 µM) with 4 µM neferine/8 µM isoliensinine induced cell death in a synergistic manner as described by isobologram. Neferine/isoliensinine could confer extensive intracellular reactive oxygen species generation in CSCs. Neferine/isoliensinine or combinatorial regimens dissipated mitochondrial membrane potential and enhanced intracellular [Ca2+ ]i, which were measured by spectroflurimetry. Furthermore, these combinatorial regimens induced a significant increase in the sub G0 phase of cell cycle arrest and PI uptake and alleviated the expression of ERCC1 in CSCs. Combinatorial regimens or neferine/isoliensinine treatments downregulated the cell survival protein expression (PI3K/pAkt/mTOR) and activated mitochondria-mediated apoptosis by upregulating Bax, cytochrome c, caspase-3, and PARP cleavage expression while downregulating the BCl-2 expression in CSCs. Our study confirms the chemosensitizing efficacy of neferine/isoliensinine or combinatorial regimens of neferine/isoliensinine with a low dose of cisplatin against CSCs.

The effect of PPARγ agonist on SGLT2 and glucagon expressions in alpha cells under hyperglycemia.

BACKGROUND: Although sodium glucose cotransporter 2 (SGLT2) inhibitors have many beneficial effects for type 2 diabetes, including decreased cardiovascular death, recent reports that they increased glucagon through SGLT2 inhibition raised some concern. Troglitazone, Peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, was reported to increase SGLT2 in renal proximal tubule cells, but its role on pancreatic alpha cells have not been reported. We investigated the effect of troglitazone on SGLT2 expression in alpha cells and subsequent glucagon regulation in hyperglycemia. METHODS: An Alpha TC1-6 cell line was cultured in control (5 mM) or hyperglycemia (HG, 15 mM) for 72 h. We applied troglitazone with or without PPARγ antagonist (GW9662 10 µM). To investigate the involvement of PI3K/Akt pathway, we applied troglitazone with or without Wortmanin. We measured sodium glucose transporter 2 (SGLT2) and glucagon (GCG) mRNA and protein expression. PPAR gamma, PI3K and Akt protein were also measured. RESULTS: Exposure of alpha TC cells to HG for 72 h increased glucagon mRNA and protein expression. HG decreased SGLT2 mRNA and protein expression. Troglitazone significantly reversed HG-induced reduction of SGLT2 expression and increase of glucagon secretion. PPARγ antagonist (GW9662 10 µM) decreased the expression of SGLT2 and increased glucagon as HG did. Hyperglycemia increased PI3K and pAkt expression in alpha cells. Wortmanin (PI3K inhibitor, 1 µM) reversed HG-induced SGLT2 decrease and glucagon increase. Troglitazone treatment decreased PI3K and pAkt expression in HG. CONCLUSION: In conclusion, PPARγ agonist, troglitazone improved glucose transport SGLT2 dysfunction and subsequent glucagon dysregulation in alpha cell under hyperglycemia. Those effects were through the involvement of PI3K/pAkt signaling pathway. This study may add one more reason for the ideal combination of PPARγ agonist and SGLT2 inhibitor in clinical practice.

Comparison of pancreatic beta cells and alpha cells under hyperglycemia: Inverse coupling in pAkt-FoxO1.

Type 2 diabetes manifests beta cell deficiencies and alpha cell expansion which is consistent with relative insulin deficiency and glucagon oversecretion. The effects of hyperglycemia on alpha cells are not as understood in comparison to beta cells. Hyperglycemia increases oxidative stress, which induces Akt activation or FoxO activation, depending on cell type. Several studies independently reported that FoxO1 translocations in alpha cells and beta cells were opposite. We compared the responses of pancreatic alpha cells and beta cells against hyperglycemia. Alpha TC-1 cells and Beta TC-6 cells were incubated with control (5mM Glucose) or high glucose (33mM Glucose) with or without PI3K inhibitor or FoxO1 inhibitor. We assessed PI3K, pAkt and phosphorylated FoxO1 (pFoxO1) in both cell lines. Immunostaining of BrdU and FoxO1 was detected by green fluorescence microscopy and confocal microscopy. Hyperglycemia and H2O2 decreased PI3K and pAKT in beta cells, but increased them in alpha cells. FoxO1 localizations and pFoxO1 expressions between alpha cells and beta cells were opposite. Proliferation of beta cells was decreased, but alpha cell proliferation was increased under hyperglycemia. Antioxidant enzymes including superoxide dismutase (SOD) and catalase were increased in beta cells and they were reversed with FoxO1 inhibitor treatment. Increased proliferation in alpha cells under hyperglycemia was attenuated with PI3K inhibitor. In conclusion, hyperglycemia increased alpha cell proliferation and glucagon contents which are opposite to beta cells. These differences may be related to contrasting PI3K/pAkt changes in both cells and subsequent FoxO1 modulation.