The effect of acute alcohol exposure on hepatic oxidative stress and function: prevention by micronutrients

Alcohol binge drinking, especially in teenagers and young adults is a major public health issue in the UK, with the number of alcohol related liver disorders steadily increasing. Understanding the mechanisms behind liver disease arising from binge-drinking and finding ways to prevent such damage are currently important areas of research. In the present investigation the effect of acute ethanol administration on hepatic oxidative damage and apoptosis was examined using both an in vivo and in vitro approach; the effect of micronutrient supplementation prior and during ethanol exposure was also studied. The following studies were performed: (1) ethanol administration (75 mmol/kg body weight) and cyanamide pre-treatment followed by ethanol to study elevated acetaldehyde levels with liver tissue analysed 2.5, 6 and 24 hours post-alcohol; (2). Using juvenile animals, 2% betaine supplementation followed by acute ethanol with tissue analysed 24 hrs post ethanol; and (3). Micronutrient supplementation during concomitant ethanol exposure to hepG2 cells. It was found that a single dose of alcohol caused oxidative damage to the liver of rats at 2.5 hr post-alcohol as evidenced by decreased glutathione levels and increased malondialdehyde levels in both the cytosol and mitochondria. Liver function was also depressed but there were no findings of apoptosis as cytochrome c levels and caspase 3 activity was unchanged. At 6 hours, the effect of ethanol was reduced suggesting some degree of recovery, however, by 24 hours, increased mitochondrial oxidative stress was apparent. The effect of elevated acetaldehyde on hepatic damage was particularly evident at 24 hours, with some oxidative changes at earlier time points. At 24 hours, acetaldehyde caused a profound drop in glutathione levels in the cytosol and hepatic function was still deteriorating. Studies examining ethanol exposure to juvenile livers showed that glutathione levels were increased, suggesting an overtly protective response not seen in with older animals. It also showed that despite cytochrome c release into the cytosol, caspase-3 levels were not increased. This suggests that ATP depletion is preventing apoptosis initiation. Betaine supplementation prevented almost all of the alcohol-mediated changes, suggesting that the main mechanism behind alcohol-mediated liver damage is oxidative stress. Results using the hepG2 cell line model showed that micronutrients involved in glutathione synthesis can protect against hepatocyte damage caused by alcohol metabolism, with reduced reactive oxygen species and increased/maintained glutathione levels. In summary, these results demonstrate that both acute alcohol and acetaldehyde can have damaging effects to the liver, but that dietary intervention may be able to protect against ethanol induced oxidative stress.

Acute hypoxia improves insulin sensitivity (SI2*) and β cell function in individuals with Type 2 Diabetes

Type 2 diabetes is a multifactorial metabolic disease characterized by defects in β-cells function, insulin sensitivity, glucose effectiveness and endogenous glucose production (1). It is widely accepted that insulin and exercise are potent stimuli for glucose transport (2). Acute exercise is known to promote glucose uptake in skeletal muscle via an intact contraction stimulated mechanism (3), while post-exercise improvements in glucose control are due to insulin-dependant mechanisms (2). Hypoxia is also known to promote glucose uptake in skeletal muscle using the contraction stimulated pathway. This has been shown to occur in vitro via an increase in β-cell function, however data in vivo is lacking. The aim of this study was to examine the effects of acute hypoxia with and without exercise on insulin sensitivity (SI2*), glucose effectiveness (SG2*) and β-cell function in individuals with type 2 diabetes. Following an overnight fast, six type 2 diabetics, afer giving informed written consent, completed 60 min of the following: 1) normoxic rest (Nor Rest); 2) hypoxic rest [Hy Rest; O2 = 14.6 (0.4)%]; 3) normoxic exercise (Nor Ex); 4) hypoxic exercise [Hy Ex; O2 = 14.6 (0.4)%]. Exercise trails were set at 90% of lactate threshold. Each condition was followed by a labelled intravenous glucose tolerance test (IVGTT) to provide estimations of SI2*, SG2* and β-cell function. Values are presented as means (SEM). Two-compartmental minimal model analysis showed SI2* to be higher following Hy Rest when comparisons were made with Nor Rest (P = 0.047). SI2* was also higher following Hy Ex [4.37 (0.48) x10-4 . min-1 (μU/ml)] compared to Nor Ex [3.24 (0.51) x10-4 . min-1 (μU/ml)] (P = 0.048). Acute insulin response to glucose (AIRg) was reduced following Hy Rest vs. Nor Rest (P = 0.014 - Table 1). This study demonstrated that 1) hypoxia has the ability to increase glucose disposal; 2) hypoxic-induced improvements in glucose tolerance in the 4 hr following exposure can be attributed to improvements in peripheral SI2*; 3) resting hypoxic exposure improves β-cell function and 4) exercise and hypoxia have an additive effect on SG2* in type 2 diabetics. These findings suggest a possible use for hypoxia both with and without exercise in the clinical treatment of type 2 diabetes.

Acute hypoxia reduces plasma myostatin independent of hypoxic dose

Background: Muscle atrophy is seen ~ 25 % of patients with cardiopulmonary disorders, such as chronic obstructive pulmonary disorder and chronic heart failure. Multiple hypotheses exist for this loss, including inactivity, inflammation, malnutrition and hypoxia. Healthy individuals exposed to chronic hypobaric hypoxia also show wasting, suggesting hypoxia alone is sufficient to induce atrophy. Myostatin regulates muscle mass and may underlie hypoxic-induced atrophy. Our previous work suggests a decrease in plasma myostatin and increase in muscle myostatin following 10 hours of exposure to 12 % O2. Aims: To establish the effect of hypoxic dose on plasma myostatin concentration. Concentration of plasma myostatin following two doses of normobaric hypoxia (10.7 % and 12.3 % O2) in a randomised, single-blinded crossover design (n = 8 lowlanders, n = 1 Sherpa), with plasma collected pre (0 hours), post (2 hours) and 2 hours following (4 hours) exposure. Results: An effect of time was noted, plasma myostatin decreased at 4 hours but not 2 hours relative to 0 hours (p = 0.01; 0 hours = 3.26 [0.408] ng.mL-1, 2 hours = 3.33, [0.426] ng.mL-1, 4 hours = 2.92, [0.342] ng.mL-1). No difference in plasma myostatin response was seen between hypoxic conditions (10.7 % vs. 12.3 % O2). Myostatin reduction in the Sherpa case study was similar to the lowlander cohort. Conclusions: Decreased myostatin peptide expression suggests hypoxia in isolation is sufficient to challenge muscle homeostasis, independent of confounding factors seen in chronic cardiopulmonary disorders, in a manner consistent with our previous work. Decreased myostatin peptide may represent flux towards peripheral muscle, or a reduction to protect muscle mass. Chronic adaption to hypoxia does not appear to protect against this response, however larger cohorts are needed to confirm this. Future work will examine tissue changes in parallel with systemic effects.

The role of acute ambient hypoxia in the regulation of myostatin

When exposed to chronic hypoxia by pathophysiological or environmental causes humans show muscle atrophy, challenging homeostasis and increasing mortality rate. Chronic hypoxia also presents with elevated myostatin peptide, a negative regulator of muscle size. This work induced acute hypoxia in healthy individuals; hypothesizing hypoxia would increase myostatin expression in both muscle and plasma in a concentration- and time-dependent manner. Hypoxia (1 % O2) reduced C2C12 myoblast migration and myotube size in vitro. Myotube atrophy was time-dependent, longer exposures showed greater atrophy. Intracellular myostatin peptide was decreased at every time point measured. Myostatin and downstream signalling pathways in muscle showed a high degree of percentage similarity between mouse and human, when amino acid sequences were directly compared. Healthy males (N = 8) were exposed to 20.9 % O2 or 11.9 % O2 for 2 hours. Following hypoxic exposure myostatin peptide was reduced in muscle but not plasma, relative to control conditions. A second cohort (N = 8) was exposed to 12.5 % O2 for 10 hours. Plasma myostatin was decreased following hypoxia, muscle myostatin trended towards increasing. A third cohort (N = 9; n = 8 lowlander, n = 1 Sherpa) was exposed to 10.7 % or 12.3 % O2 for 2 hours. Plasma myostatin was reduced at both concentrations with no difference between concentrations noted. In response to chronic hypoxia, individuals lose muscle mass. Counter to the hypothesis of an increase in myostatin in both muscle and plasma, here a consistent decrease in plasma myostatin following acute hypoxia is seen. Muscle myostatin shows a variable response, with decreasing intracellular expression seen following a 2 hour hypoxic exposure, and trends towards an increase following 10 hours of hypoxia. Decreases in plasma and muscle myostatin may represent myostatin’s movement towards peripheral compartments in these acute timeframes. Hypoxia alone is capable of altering myostatin in healthy individuals; the effects of hypoxia on myostatin appear to differ between the acute timeframes examined here and chronic exposures in environmental or disease models.