Different origin of hypertriglyceridemia induced by a high-fat and a high-sucrose diet in ventromedial hypothalamic-lesioned obese and normal rats.

OBJECTIVE: To clarify the mechanism by which plasma triacylglycerol is affected by a high fat or a sucrose diet. DESIGN: Two sets of six groups each having six rats were prepared-(1) ventromedial hypothalamic (VMH)-lesioned rats fed a standard diet; (2) sham VMH-lesioned rats fed a standard diet; (3) VMH-lesioned rats fed a high-fat diet; (4) sham VMH-lesioned rats fed a high-fat diet; (5) VMH-lesioned rats fed a high-sucrose diet; and (6) sham VMH-lesioned rats fed a high-sucrose diet. After VMH lesions and sham operations, the rats were provided standard, high-fat and high sucrose diets for 2 weeks. Two weeks later, blood samples were collected after overnight fast to determine plasma triacylglycerol (TAG), hepatic triacylglycerol secretion rate (TGSR), fractional catabolic rate (FCR) of triacylglycerol and postheparin plasma lipoprotein lipase (LPL), plasma glucose, insulin and leptin. RESULTS: Values of TAG, TGSR, FCR and LPL in VMH-lesioned obese rats were all greater than those in sham-operated rats, regardless of the diet fed. In sham-operated rats, high-fat diet fed rats showed higher TAG with similar TGSR, higher LPL and lower FCR than those of standard diet fed rats. High-sucrose diet fed rats showed significantly higher TAG with higher TGSR, higher LPL and lower FCR than those of standard diet fed rats. Moreover, high-sucrose diet fed rats showed higher TAG with higher TGSR, lower LPL and higher FCR than those of high-fat diet fed rats. In VMH-lesioned rats, high-fat diet fed rats showed higher TAG with similar TGSR, higher LPL and lower FCR than those of standard diet fed rats. High-sucrose diet fed rats showed markedly higher TAG with notably higher TGSR, higher LPL and lower FCR than those of standard diet fed rats. High-sucrose diet fed rats showed still higher TAG with markedly higher TGSR, similar LPL and higher FCR than those of high-fat diet fed rats. CONCLUSIONS: The mechanism by which TAG metabolism is affected by a high-fat or a high-sucrose diet differed; a high-fat diet increased plasma TAG level by lowering removal of TAG without increase in hepatic TAG secretion in sham-operated (normal) rats. A high-sucrose diet, in contrast, induced much higher plasma TAG levels by both increased hepatic TAG secretion and decreased removal of TAG. The effects of a high-fat or a high-sucrose diet were similar but exaggerated in VMH lesioned animals.

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.

Impaired reductive regeneration of ascorbic acid in the Goto-Kakizaki diabetic rat.

Ascorbic acid (AA) is a naturally occurring major antioxidant that is essential for the scavenging of toxic free radicals in both plasma and tissues. AA levels in plasma and tissues have been reported to be significantly lower than normal in diabetic animals and humans, and might contribute to the complications found at the late stages of diabetes. In this study, plasma and hepatic AA levels and AA regeneration were studied in the Goto-Kakizaki diabetic rat (GK rat) to elucidate the mechanism of decreasing plasma and hepatic AA levels in diabetes. AA concentrations in the plasma and liver were significantly lower in GK than in control rats. AA levels in primary cultured hepatocytes derived from GK rats were lower than those derived from control Wistar rats with or without dehydroascorbic acid (DHA) in the medium. Among various enzyme activities that reduce DHA to AA, the NADPH-dependent regeneration of AA in the liver was significantly suppressed in GK rats. Northern blot analysis revealed that only the expression of 3-alpha-hydroxysteroid dehydrogenase (AKR) was significantly suppressed in these rats. These results suggest that decreased AA-regenerating activity, probably through decreased expression of AKR, contributes to the decreased AA levels and increased oxidative stress in GK rats.

Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form.

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.

Nitric oxide-independent effects of nitric oxide donors on energy metabolism in erythrocytes.

In order to study the roles of nitric oxide (NO) in various biological events, several types of NO-releasing agents have been extensively used. Although both NO and its donors and/or their decomposed products may have biological activities, most of the cellular responses to these donors have been postulated to reflect NO-dependent events. Among the various NO donors, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-l-triazene (NOC7), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), S-nitrosoglutathione, S-nitrosocysteine (CysNO), and related nitrosothiols are commonly used agents. To investigate the biological activities of these donors and their decomposed products, we tested their effects on energy metabolism in erythrocytes. When incubated with freshly prepared erythrocytes, NOC7, Cys-NO, and their decomposed products, but not NO and its oxidized metabolites, nitrite and nitrate, decreased cellular ATP levels. Although SIN-1 generates both NO and superoxide radical thereby forming peroxynitrite (ONOO-), this donor had no appreciable effect on cellular ATP levels, even in the presence of superoxide dismutase. These results indicate that NOC7 and CysNO and/or their decomposed product(s), but not NO and its oxidized metabolites, are responsible for the decrease in cellular ATP levels. Thus, the effects of not only NO and its oxidized metabolites (NO2, NO3 ), but also NO donors and their decomposed products, should be taken into account when attempting to understand the mechanism of biological responses induced by NO donors.

Role of glutathione in stabilization of nitric oxide during hypertension developed by inhibition of nitric oxide synthase in the rat.

The present study examined the role of glutathione in the development of hypertension induced by long-term inhibition of nitric oxide (NO)-synthase. Three groups of rats were investigated: control group, L-NAME group: group with NO-synthase inhibition by N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg per day) for 2 weeks, and BSO group: group with glutathione synthesis inhibitor L-buthionine sulfoximine (BSO, 1.4 mmol/kg per 12 h) for 3 days. All the groups were subjected to an acute i.v. experiment in which the given substances were exchanged between groups. There was no change in systolic blood pressure (SBP) in the control group after 1 and 2 h of acute BSO (1.4 mmol/kg, i.v.) treatment. In the L-NAME group, SBP increased significantly by 10% after 2 h of acute BSO treatment. In the BSO group, SBP did not change vs control; however, after 2 h of acute L-NAME (10 mg/kg, i.v.) treatment, the increase in SBP exceeded by 12% (P<0.05) that of the control group. Along with the increase in SBP, acute BSO treatment significantly potentiated the decrease in plasma nitrite/nitrate concentration in the L-NAME group. The acute BSO-induced glutathione decrease was significantly greater in the L-NAME group than in the control group. In NO-deficient hypertensive rats, the results are indicative of a decrease in glutathione synthesis and a stabilizing role of glutathione.

Rapid increase in circulating leptin in ventromedial hypothalamus-lesioned rats: role of hyperinsulinemia and implication for upregulation mechanism.

The mechanisms of marked increase in plasma leptin soon after ventromedial hypothalamus (VMH) lesions were investigated. Although rats did not gain body weight or parametrial fat-pad mass 24 h after the operation, the acute VMH-lesioned rats exhibited substantial five- and fourfold increases in plasma leptin levels compared with sham-operated control rats in fed (22.6 +/- 3.2 vs. 5.8 +/- 1.2 ng/ml) and fasted (8.8 +/- 2.0 vs. 2.3 +/- 0.3 ng/ml) states, respectively. Plasma insulin concentration was doubled in VMH-lesioned rats compared with sham-operated controls in both fed and fasting states. Northern blot analysis revealed that mRNA of ob gene was not increased in parametrial fat pad of animals 24 h after the creation of VMH lesions. However, leptin content in the fat pad was significantly increased in VMH-lesioned rats compared with sham-operated controls (32.2 +/- 4.7 vs. 17.4 +/- 2.3 ng/g wet tissue). The leptin content in parametrial fat pad was highly correlated with plasma leptin concentrations (r = 0.898, P < 0.001). To define the effect of hyperinsulinemia on their hyperleptinemia, a small dose of streptozotocin (STZ) (25 mg/kg body wt) was intravenously administered into rats 5 days before the creation of VMH lesions. Plasma insulin levels were not increased after VMH lesions in STZ-pretreated rats. Plasma leptin levels were halved in the absence of hyperinsulinemia, but still remained twofold higher than those in their sham-operated counterparts (9.9 +/- 1.3 vs. 4.8 +/- 0.7 ng/ml). These results indicate that the destruction of VMH rapidly promotes leptin production before obesity develops through an enhanced translational process in which hyperinsulinemia occurring after VMH lesioning plays an important role. The present study also suggests that there are other mechanisms that rapidly upregulate leptin production in adipocytes in VMH-lesioned rats in which the target organ of this hormone has been destroyed.

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life.

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H+ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.

Fates and vascular action of S-nitrosoglutathione and related compounds in the circulation.

To know the metabolism of low-molecular-weight S-nitrosothiols (RS-NO) in the circulation, we analyzed the stability and depressor effects of S-nitrosoglutathione (GS-NO) and the l- and d-forms of S-nitrosocysteine (Cys-NO). Although half-lives of these RS-NO in fresh plasma were longer than 50 min, their depressor effects disappeared within 5 min after intravenous administration of these compounds. Acivicin (AT-125), an inhibitor of gamma-glutamyltransferase (gamma-GTP), prolonged the depressor effect of GS-NO but not of Cys-NO. The depressor effect of GS-NO disappeared in AT-125-treated rats within 10 min after administration, which is still shorter than its half-life in vitro. Although S-conjugates of l-cysteine, but not of d-cysteine, rapidly enter into cells via an active transport system and disappear from the circulation, both forms of Cys-NO exhibited similar activity to decrease blood pressure to that of NO. Thus, NO might be rapidly released from Cys-NO in the circulation and shortly exhibited its depressor action. These observations suggested that the circulating GS-NO is rapidly decomposed by gamma-GTP to form Cys-NO and that the release of NO from both GS-NO and Cys-NO is enhanced significantly in the circulation.

Helicobacter pylori generates superoxide radicals and modulates nitric oxide metabolism.

During studies of the bactericidal action of nitric oxide (NO), we found that it reversibly inhibited the respiration of Escherichia coli and irreversibly inhibited the respiration of Helicobacter pylori. Peroxynitrite, a reaction product of NO and superoxide, irreversibly inhibited the respiration of both H. pylori and E. coli. H. pylori, but not E. coli, generated substantial amounts of superoxide radicals. These results suggest that NO directly inhibits the respiration of E. coli whereas it rapidly reacts with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivates the respiration of H. pylori.

Role of glutathione in nitric oxide-dependent regulation of energy metabolism in rat hepatoma cells.

Previous studies in this laboratory revealed that nitric oxide (NO) reversibly inhibits the respiration of isolated mitochondria and ascites hepatoma (AH-130) cells by an oxygen concentration-dependent mechanism. The inhibitory effect of NO on the respiration of AH-130 cells was enhanced by treating with digitonin that selectively permeabilized plasma membranes and released cytosolic low-molecular-weight compounds. Reduced glutathione (GSH) is the most abundant cytosolic thiol that easily reacts with NO. To elucidate the mechanism by which digitonin enhanced the inhibitory action of NO, the effect of GSH and related thiols was studied with AH-130 cells and their mitochondria. The inhibitory effect of NO on the respiration of digitonin-treated cells was suppressed by either GSH, L-cysteine, or N-acetylcysteine, but not by oxidized glutathione. The inhibitory effect of NO on the respiration of their mitochondria was also decreased by GSH. In contrast, the inhibitory effect of NO was markedly enhanced with AH-130 cells obtained from animals that were pretreated with L-buthionine sulfoximine (BSO), a specific inhibitor for GSH synthesis. Kinetic analysis revealed that NO dose-dependently decreased GSH levels in AH-130 cells with concomitant generation of S-nitrosothiols. Although S-nitrosoglutathione (GSNO), a slow releaser of NO, also inhibited the respiration of tumor cell mitochondria, its effect was significantly lower than that of NO. These results suggest that cellular GSH might play pivotal roles in the regulation of energy metabolism in hepatoma cells by modulating free forms of NO.

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats.

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.

Oxygen-dependent regulation of the respiration and growth of Escherichia coli by nitric oxide.

To elucidate the role of nitric oxide (NO) in the metabolisms of enteric bacteria, its effect on the respiration and growth of Escherichia coli was examined. Respiration of E. coli was reversibly inhibited by NO particularly under low oxygen tensions. Growth of E. coli was also inhibited by NO more strongly under low oxygen tension than at its high concentration. Because the intestinal lumen is anaerobic, even a small amount of NO might strongly inhibit the energy metabolism and growth of E. coli and other enteric bacteria in vivo than in air atmospheric conditions in which oxygen tension is unphysiologically high.