Ichiro Sonaka

Identification and characterization of oxidized human serum albumin

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases. However, little is known regarding the pathophysiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both the structural and functional differences between reduced and oxidized HSA. Using LC‐ESI‐TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide‐bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand‐binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions.

Leucine promotes glucose uptake in skeletal muscles of rats

Soleus muscles isolated from normal rats were incubated to evaluate whether or not leucine promotes glucose uptake under insulin-free conditions, using a labeled 2-deoxyglucose uptake assay. Glucose uptake was promoted by 2mM leucine. A metabolite of leucine, alpha-ketoisocaproic acid (alpha-KIC), also exhibited a similar stimulatory effect, although this was not as potent as leucine. Stimulation of glucose uptake by leucine was completely canceled by pre-treatment with either 10 microM LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), or 6 microM GF109203X, a specific inhibitor of protein kinase C (PKC). No significant change was observed by pre-treatment with 1 microM rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR). These results suggest that leucine stimulates glucose transport in skeletal muscle via PI3-kinase and PKC pathways independently of the mammalian target of mTOR. They also suggest that leucine stimulates glucose transport by an insulin-independent mechanism.

L-Cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.

An elemental diet controls inflammation in indomethacin-induced small bowel disease in rats: the role of low dietary fat and the elimination of dietary proteins.

Elemental diets (EDs) are effective in treating Crohns disease. We hypothesize that low dietary fat and amino acids used as the sole nitrogen source are the major contributors for the success of EDs. We examined the influences of the addition of dietary fat and protein to an ED using an indomethacin-induced inflammation model in rat small intestine. In the ED-fed rats, the intestinal damage score was decreased compared with that in the standard chow group with decreasing intestinal permeability. By supplementing an ED with soybean oil (SO), intestinal permeability was increased to a level similar to that of the standard chow group. For this group, the intestinal damage score also increased compared with that of the ED group but did not reach the levels observed in the standard chow group.The addition of dietary proteins (using heat-denatured pancreatin) resulted in intestinal damage scores that were significantly higher than those of the ED+SO-fed group. The dietary protein increased the intestinal damage score.These results suggest that EDs control inflammation by decreasing intestinal permeability and the elimination of dietary proteins.

Redox state of albumin is not associated with colloid osmotic pressure

Serum albumin exists in oxidized and reduced forms. Although the oxidation of albumin affects some of its functions, the relationship between oxidized albumin and colloid osmotic pressure (COP) remains unclear. The aim of this study was to determine whether there is an association between oxidized albumin and COP. Blood samples from 20 healthy volunteers were divided into two aliquots in order to prepare reduced (n=20) and oxidized albumin samples (n=20). This was achieved by treatment with L-cysteine and a redox-stabilizing agent before and after incubation at 37°C for 24 h. The percentage of oxidized albumin was determined by high-performance liquid chromatography. COP was measured using a colloid osmometer. Reduced and oxidized albumin samples showed 100% of reduced and 100% of oxidized albumin, respectively. There were no significant differences in albumin level and total protein level between the reduced and the oxidized albumin samples. No significant change was seen in COP between the reduced and the oxidized albumin samples (reduced albumin, 17.4±0.2 mmHg; oxidized albumin, 17.3±0.2 mmHg; P=0.465). Therefore, there is no significant difference in COP between reduced and oxidized albumin samples.

Branched-chain amino acids inhibit the TGF-beta-induced down-regulation of taurine biosynthetic enzyme cysteine dioxygenase in HepG2 cells.

Taurine deficiency has been suggested to contribute to the pathogenesis and complications of advanced hepatic diseases. The molecular basis for a low level of taurine associated with hepatic failure is largely unknown. Using carbon tetrachloride (CCl4)-induced cirrhotic rat model, we found that the activity and expression of cysteine dioxygenase (CDO), a rate-limiting enzyme in taurine synthesis, were significantly decreased in the liver of these rats. To investigate the underlying mechanisms for the suppression, we examined the effects of pathological cytokines on CDO expression in human hepatoma HepG2 cells. Among the several cytokines, transforming growth factor-β (TGF-β), one of the key mediators of fibrogenesis, suppressed Cdo1 gene transcription through the MEK/ERK pathway. Finally, we further examined potential effects of branched-chain amino acids (BCAA) on CDO expression, as it has been reported that oral BCAA supplementation increased plasma taurine level in the patients with liver cirrhosis. BCAA, especially leucine, promoted Cdo1 gene transcription, and attenuated TGF-β-mediated suppression of Cdo1 gene expression. These results indicate that the low plasma level of taurine in advanced hepatic disease is due to decreased hepatic CDO expression, which can be partly attributed to suppressive effect of TGF-β on Cdo1 gene transcription. Furthermore, our observation that BCAA promotes Cdo1 expression suggests that BCAA may be therapeutically useful to improve hepatic taurine metabolism and further suppress dysfunctions associated with low level of taurine in hepatic diseases.