The Synergistic Action of Chronic Ethanol and Reactive Oxygen Species on Insulin Signaling in Hypertensive Vascular Smooth Muscle Cells

Chronic Ethanol (ETOH) and Reactive Oxygen Species (ROS) have been shown to have an effect on insulin signaling in Hypertensive Vascular Smooth Muscle Cells (HVSMCs). The hypothesis was tested if insulin signaling is altered by increases in oxidative stress due to ETOH exposure in HVSMCs using MAPKs as an indicator. HVSMCs were treated with increased concentrations of ETOH (1-16 mM), and 1000 µM H2O2, and stimulated with 8 µM insulin with maximum activation of ERK 1/2 occurring with 8 mM ETOH. Exposing HVSMCs to 8 mM ETOH and H2O2 1-40 min then 8 µM insulin resulted in maximum activation of ERK 1/2 within 1-10 minutes. The data indicate that Akt was inhibited between 1-10 min in both hypertensive and normal cells. RT-PCR analysis shows that Nox 1 expression in HVSMCs increased in a timely manner at 1-10 min and decreased at 20-40 min when cells were treated with ETOH for 5 min. In normal cells Nox 1 expression was not as dramatic as what was observed in HVSMCs. In a second set of experiments using mRNA isolated with Nox 4 indicated that Nox 4 mRNA expression increased at 1 min and decreased at 5 min and a dramatic increase in expression for 10-40 min. In normal cells the observable reduction was observed at 10 min. Real-time PCR quantitative analysis of the data indicated that between 1-5 min Nox 1 decreased significantly with a timely increase at 10-40 min of treating cells with ETOH. Again, this quantitative profile was observed with Nox 4. FoxO3 in hypertensive cells compared to control expresses an increase until 40 min of ETOH exposure, but in normal cells a decrease is evident beginning at 20 minutes. With FoxO4 in hypertensive cells there is a gradual increase in expression compared to control and significant increase at 10, 20 and 40 minutes after ETOH exposure. In normal cells, FoxO4 increases compared to control at 1 and 5 minutes, but a significant decrease is observed at 10, 20 and 40 minutes. The data suggests that the effect of ETOH alters insulin signaling by increasing oxidative stress in both cell types

Chronic ethanol exposure alters expression of insulin-induced mitogen-activated protein kinases in hypertensive vascular smooth muscle cells

Insulin resistance is an important risk factor in development of cardiovascular diseases such as hypertension and atherosclerosis. However, the specific role of insulin resistance in the etiology of these diseases is poorly understood. On the other hand, ethanol is a potent vasoconstrictor which induces changes in mitogen activated protein kinases. We hypothesize that in hypertensive vascular smooth muscle cells (VSMCs), ethanol (ETOH) interferes with insulin action by altering mitogen activated protein kinases, the major signaling molecules implicated in the biological actions of insulin. By stimulating hypertensive (SHR) cells with 1–16 µM of insulin, ERK 1 & 2 expression increased in a time dependent manner with maximum expression occurring at 10 minutes, whereas, in normal cells (WKY), no significant increase in expression of ERK 1 & 2 was observed for at least 20 minutes. We measured expression of AKT under similar conditions. By contrast, in hypertensive cells, insulin inhibited AKT expression within the first 5 minutes. This observed insulin-induced inhibition of AKT was not observed at 10, 20, and 40 min of stimulation. On the contrary, exposing hypertensive cells chronically (24 hr) to elevated concentrations (50–800 mM) of ethanol prior to stimulating with insulin, ERK 1 & 2 expression decreased in a biphasic manner with 100 and 400 mM. Eight hundred micromoles of ethanol had maximum effect. Similarly, insulin-induced AKT decreased in hypertensive cells with maximum ETOH concentrations of 400 and 800 mM. From these data, we conclude that chronic ETOH negatively alters insulin signaling in hypertensive vascular smooth muscle cells providing an alternative molecular mechanism that may increase the risk of insulin resistance. This increased risk of insulin resistance may increase the possibility of cardiovascular diseases

Alcohol Modulates the Biogenesis and Composition of Microglia-Derived Exosomes

Exosomes are small extracellular vesicles that have emerged as an important tool for intercellular communication. In the central nervous system, exosomes can mediate glia and neuronal communication. Once released from the donor cell, exosomes can act as discrete vesicles and travel to distant and proximal recipient cells to alter cellular function. Microglia cells secrete exosomes due to stress stimuli of alcohol abuse. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from microglia cell line BV-2. The BV-2 cells were cultured in exosome-free media and were either mock treated (control) or treated with 50 mM or 100 mM of alcohol for 48 and 72 h. Our results demonstrated that alcohol significantly impacted BV-2 cell morphology, viability, and protein content. Most importantly, our studies revealed that exosome biogenesis and composition was affected by alcohol treatment