Expression of hepatic antioxidant enzymes in non-obese type-2 diabetic Goto-Kakizaki rats.

Diabetes mellitus and its complications have been attributed in part to oxidative stress, against which antioxidant enzymes constitute a major protective mechanism. The present study was performed to investigate the effects of early stage type 2 diabetes in the absence of obesity and liver damage on hepatic antioxidant enzyme expression and oxidative stress using 9-week-old Goto-Kakizaki (GK) rats. Hepatic total antioxidant capacity determined by total oxygen radical scavenging capacity and lipid peroxidation determined by malondialdehyde in plasma and liver were not significantly different between normal Wistar rats and GK rats. These results indicated that oxidative stress is not evident in these type 2 diabetic rats. Hepatic expression levels of antioxidant enzymes, including superoxide dismutase-1, catalase, glutathione peroxidase and reductase, thioredoxin-1, mu- and pi-class glutathione S-transferase (GST), and the gamma-glutamylcysteine ligase catalytic subunit, were not different between normal rats and GK rats. But, hepatic level and activity of alpha-class GST were decreased and peroxiredoxin-1 level was increased in GK rats, suggesting that upregulation of peroxiredoxin-1 compensates for downregulation of alpha-class GST. These results suggest that alpha-class GST and peroxiredoxin-1 in liver can be altered during the early stages of type 2 diabetes in the absence of obesity and severe oxidative stress.

Alterations in hepatic metabolism of sulfur amino acids in non-obese type-2 diabetic Goto-Kakizaki rats.

Elevated plasma homocysteine has been identified as a risk factor for cardiovascular disease and non-alcoholic liver disease, which are major complications of diabetes. Hence, hepatic homocysteine metabolism has become a major focus of diabetes research. However, little information is available regarding plasma homocysteine levels in non-obese diabetic animals. Therefore, we investigated the hepatic metabolism of sulfur-amino acids in non-obese type-2 diabetic Goto-Kakizaki rats. The experiments were performed using 9-week-old Goto-Kakizaki rats and age-matched Wistar rats. The major finding of this study is that homocysteine levels in the liver and plasma are maintained by a balance between the up-regulation of betaine homocysteine methyltransferase and the inhibition of cystathionine ß-synthase in non-obese type-2 diabetic rats. Hepatic levels of cysteine and its metabolites, such as hypotaurine, taurine, and glutathione, were increased despite inhibition of the transsulfuration of homocysteine to cysteine. The elevated hepatic taurine and glutathione levels may be attributed to the up-regulation of cysteine dioxygenase expression and increased cysteine availability for glutathione synthesis. Inhibition of hepatic methionine adenosyltransferase activity in Goto-Kakizaki rats was associated with a decrease in hepatic S-adenosylmethionine, which serves as an allosteric activator of cystathionine ß-synthase. The non-obese type-2 diabetic condition results in profound changes in hepatic sulfur-amino acid metabolism and raises the possibility that sulfur-amino acid metabolism may be regulated by obesity- as well as diabetes-associated factors. Further study to elucidate the pathological significance of sulfur-amino acid metabolism in chronic liver disease in type-2 diabetic animals is underway in this laboratory.

Plasma homocysteine level and hepatic sulfur amino acid metabolism in mice fed a high-fat diet.

PURPOSE: Obesity, a feature of metabolic syndrome, is a risk factor for cardiovascular disease, and elevated plasma homocysteine is associated with increased cardiovascular risk. However, little published information is available concerning the effect of obesity on homocysteine metabolism. METHODS: Hepatic homocysteine metabolism was determined in male C57BL/6 mice fed a high-fat diet for 12 weeks. RESULTS: High-fat diet increased plasma homocysteine but decreased hepatic homocysteine levels. Hepatic S-adenosylhomocysteine hydrolase levels were down-regulated in the obese mice, which was in part responsible for the decrease in hepatic S-adenosylmethionine/S-adenosylhomocysteine, which served as an index of transmethylation potential. Despite the decrease in hepatic cysteine, hepatic taurine synthesis was activated via up-regulation of cysteine dioxygenase. Hepatic levels of methionine adenosyltransferase I/III, methionine synthase, methylene tetrahydrofolate reductase, and gamma-glutamylcysteine ligase catalytic subunit were unchanged. Obese mice showed elevated betaine-homocysteine methyltransferase and decreased cystathionine beta-synthase activities, although the quantities of these enzymes were unchanged. CONCLUSION: This study suggests that plasma homocysteine level is increased in obesity-associated hepatic steatosis, possibly as a result of increased hepatic homocysteine efflux along with an altered sulfur amino acid metabolism.

Hepatic metabolism of sulfur amino acids in db/db mice.

To determine the effect of type-2 diabetes and obesity on the hepatic metabolism of sulfur amino acids, hepatic sulfur amino acid metabolism was determined in db/db mice. Hepatic methionine was markedly decreased in db/db mice, although the hepatic activity of betaine homocysteine methyltransferase was increased. The decrease in hepatic methionine was reflected by decreased sulfur-containing methionine metabolites, including S-adenosylmethionine, homocysteine, cysteine, and hypotaurine in liver and plasma. In contrast, S-adenosylhomocysteine, putrescine, and spermidine were increased in db/db mice. The hepatic level and activity of methionine adenosyltransferase I/III, an S-adenosylmethionine synthesizing enzyme, were significantly increased. These results suggest that increased polyamine synthesis, in conjunction with decreased hepatic methionine levels, is partly responsible for the reduction in hepatic S-adenosylmethionine. Decreased homocysteine in liver and plasma may be attributable to the decrease in hepatic methionine and upregulation of hepatic betaine homocysteine methyltransferase. Glutathione in liver and plasma did not change despite decreased γ-glutamylcysteine ligase activity. The decreased hepatic hypotaurine may be attributable to the downregulation of cysteine dioxygenase. The major finding of this study is that db/db mice exhibited decreases in hepatic methionine and its sulfurcontaining metabolites.

Hepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats.

Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto-Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and ß-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b(5), CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels.

Expression of hepatic and ovarian cytochrome P450 during estrous cycle in rats.

It is known that gender differences in drug metabolism are largely attributed to changes in sex and growth hormones. Serum concentrations of estradiol, progesterone, prolactin, follicle-stimulating hormone, and luteinizing hormone change markedly during the human menstrual cycle and the rat estrous cycle. However, little information is available regarding the effects of the human menstrual cycle or the rat estrous cycle on expression and activity of cytochrome P450 (CYP) isoforms. The present study was carried out to determine the expression and activity of CYP-dependent drug-metabolizing enzymes in the liver and ovary during the estrous cycle. The expression and activity of microsomal CYP isoforms (CYP1A1, CYP1A2, CYP1B1, CYP2B1, CYP2C11, CYP2C12, CYP2E1, CYP3A1, CYP3A2, and CYP4A), cytochrome b(5) and NADPH-dependent CYP reductase in the liver and ovary were measured in female rats in diestrus and proestrus. Our results indicated that hepatic and ovarian expression and activity of CYP isoforms, cytochrome b(5), and NADPH-dependent CYP reductase were not different between diestrus and proestrus, although serum estradiol concentration and uterus weight were markedly increased in the proestrus phase. These results suggest that the cytochrome P450-dependent system is not sensitive to changes in the estrous cycle, and further studies are warranted to determine the effects of the estrous cycle on in vivo metabolism of xenobiotics.

Age-related changes in hepatic expression and activity of cytochrome P450 in male rats.

Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.