ABSTRACT
Insulin resistance is a typical precursor and primary feature of type 2 diabetes mellitus (T2DM). Sphingomyelin (SM) is a kind of sphingolipid located in animal brain, liver, kidney and muscle. Sphingomyelin synthase 2 (SMS2) is the key enzyme in the synthesis of sphingomyelin, inhibition of which shows protective effects on cardiovascular and glucose metabolism. We used Ly93, a selective sphingomyelin synthase 2 inhibitor, to investigate the effect of SMS2 inhibitor on insulin resistance in vitro and in vivo. Our previous studies have shown that Ly93 is able to dose-dependently inhibit the SMS activity and attenuate the atherosclerotic lesions in apoE knock out mice. In this present study, we found that high fat diet (HFD) induced insulin-resistant C57BL/6 mice treated with Ly93 were more sensitive to insulin than untreated mice, and presented lower blood insulin levels and improved insulin tolerance. Furthermore, insulin signal pathway related protein levels were detected by western blot, which indicated that SMS2 inhibitor significantly upregulated the phosphorylation of IRS-1, Akt and GSK-3ß, thus enhanced the insulin signaling. In vitro, Ly93 enhanced the phosphorylation of Akt in HepG2 cells, which was reversed by exogenous sphingomyelin. These results suggest that SMS2 inhibitor could ameliorate insulin resistance via regulating the insulin signaling. Our findings support that SMS2 is a potential target for insulin resistance.
Subject(s)
Enzyme Inhibitors/pharmacology , Insulin Resistance , Insulin/blood , Membrane Proteins/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Organic Chemicals/pharmacology , Transferases (Other Substituted Phosphate Groups)/antagonists & inhibitors , Animals , Diet, High-Fat , Glycogen Synthase Kinase 3 beta/metabolism , Hep G2 Cells , Humans , Insulin Receptor Substrate Proteins/metabolism , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Sphingomyelins/metabolism , Transferases (Other Substituted Phosphate Groups)/metabolismABSTRACT
Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1ß were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1ß expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1ß-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1ß-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1ß-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1ß-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.
Subject(s)
E-Selectin/metabolism , Endothelium, Vascular/drug effects , Indican/toxicity , Interleukin-1beta/agonists , MAP Kinase Signaling System/drug effects , Poisons/toxicity , Up-Regulation/drug effects , Acute Kidney Injury/blood , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Acute Kidney Injury/physiopathology , Aged , Animals , Biomarkers/blood , Biomarkers/metabolism , Cells, Cultured , Disease Models, Animal , E-Selectin/chemistry , E-Selectin/genetics , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Female , Human Umbilical Vein Endothelial Cells , Humans , Indican/blood , Interleukin-1beta/metabolism , Kidney/blood supply , Kidney/metabolism , Kidney/pathology , Kidney/physiopathology , Male , Mice, Inbred C57BL , Oxidative Stress/drug effects , Poisons/blood , Reperfusion Injury/blood , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Uremia/etiologyABSTRACT
Renal ischemia rapidly mobilizes endothelial progenitor cells (EPCs), which provides renoprotection in acute kidney injury (AKI). Indoxyl sulfate (IS) is a protein-binding uremic toxin with a potential role in endothelial injury. In this study, we examined the effects and mechanisms of action of IS on EPCs in AKI. Forty-one consecutive patients (26 male; age, 70.1 ± 14.1 years) diagnosed with AKI according to the AKIN criteria were enrolled. The AKI patients had higher serum IS levels than patients with normal kidney function (1.35 ± 0.94 × 10(-4)M vs. 0.02 ± 0.02 × 10(-4)M, P < 0.01). IS levels were negatively correlated to the number of double-labeled (CD34(+)KDR(+)) circulating EPCs (P < 0.001). After IS stimulation, the cells displayed decreased expression of phosphorylated endothelial nitric oxide (NO) synthase, vascular cell adhesion molecule-1, increased reactive oxygen species, decreased proliferative capacity, increased senescence and autophagy, as well as decreased migration and angiogenesis. These effects of IS on EPCs were reversed by atorvastatin. Further, exogenous administration of IS significantly reduced EPC number in Tie2-GFP transgenic mice and attenuated NO signaling in aortic and kidney arteriolar endothelium after kidney ischemia-reperfusion injury in mice, and these effects were restored by atorvastatin. Our results are the first to demonstrate that circulating IS is elevated in AKI and has direct effects on EPCs via NO-dependent mechanisms both in vitro and in vivo. Targeting the IS-mediated pathways by NO-releasing statins such as atorvastatin may preempt disordered vascular wall pathology, and represent a novel EPC-rescued approach to impaired neovascularization after AKI.
Subject(s)
Acute Kidney Injury/drug therapy , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Heptanoic Acids/pharmacology , Indican/toxicity , Pyrroles/pharmacology , Stem Cells/drug effects , Aged , Aged, 80 and over , Animals , Apoptosis/physiology , Atorvastatin , Blotting, Western , Centrifugation, Density Gradient , Endothelial Cells/physiology , Female , Flow Cytometry , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Indican/blood , Male , Mice , Mice, Inbred C57BL , Middle Aged , Nitric Oxide Synthase Type III/metabolism , Reactive Oxygen Species/metabolism , Stem Cells/physiology , Taiwan , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Asarones (alpha-asarone and beta-asarone) are the active components in the traditional Chinese medicine (TCM) of Acorus tatarinowii Schott, which has been used to treat epilepsy for several thousand years. To perform the pharmacokinetics (PK) study of alpha- and beta-asarone from the TCM essential oil, a simple, rapid and sensitive method was developed for the determination of asarones from the TCM in rabbit plasma, based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) with electron ionization (EI). The extraction parameters of headspace volume, fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized. Furthermore, the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 microg/mL, R(2) > 0.99), low detection limit (<2.0 ng/mL) and good precision (RSD < 7.0%). Finally, HS-SPME followed by GC/MS was applied to fast determination of alpha- and beta-asarone in rabbit plasma at different time points after oral adminstration of the essential oil from A. tatarinowii. The experimental results suggest that the proposed method provides an alternative approach to the PK studies of volatile compounds in TCMs.
