Heat stress prevents the decrease in succinate dehydrogenase activity in the extensor digitorum longus of streptozotocin-induced diabetic rats.

This study aimed to investigate whether heat stress (HS) prevents a decrease in succinate dehydrogenase (SDH) activity and heat shock protein 60 (HSP60) and superoxide dismutase 2 (SOD2) contents in the extensor digitorum longus of streptozotocin (STZ)-induced diabetic rats. Twelve-week-old male Wistar rats were assigned to one of the four groups (n=6/group): control (Con), HS, diabetes mellitus (DM), and diabetes mellitus and heat stress (DM+HS). Diabetes was induced by the administration of STZ (50 mg/kg). HS was initiated 7 days after STZ treatment and performed at 42 °C for 30 min 5 times a week for 3 weeks. SDH activity was decreased in the DM and DM+HS groups. However, SDH activity was greater in the DM+HS group than in the DM group. Although HSP60 content was lower in the DM group than in the Con group, it was maintained in the DM+HS groups and was higher than that in the DM group. SOD2 content was decreased only in the DM group. These findings suggest that HS prevents the decrease in SDH activity in the skeletal muscle induced by DM. According to this mechanism, the maintenance of SOD2 and HSP60 by HS may suppress the increase in oxidative stress.

Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats.

Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

Inflammatory responses to lipopolysaccharide are suppressed in 40% energy-restricted mice.

To elucidate the suppressive effects of energy restriction on the inflammatory responses to lipopolysaccharide (LPS), mice were divided into a control group (fed 5.0 g diet/d; 71 kJ/d) and a 40% energy-restricted group (fed 3.0 g diet/d; 43 kJ/d) at 8-wk of age. Four weeks later, 25 microg of LPS was intraperitoneally injected. After the LPS injection, interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha were elevated in serums in the 40% energy-restricted mice and in the controls, but the extent of the elevation was significantly lower in the restricted group. The LPS-induced expression of inducible nitric oxide synthase in the liver was significantly suppressed by the energy restriction. In addition, the LPS-induced elevations of serum aspartate and alanine aminotransferase activities, which are indexes of hepatic injury, were also significantly attenuated in the restricted group. Moreover, the extent of LPS-induced alterations in hepatic structure was less in the restricted mice than in controls. Serum corticosterone level in the restricted mice was higher than that in the controls before LPS treatment (P < 0.05). Furthermore, after LPS injection, the significantly higher level of corticosterone was maintained in the restricted mice, although the LPS treatment significantly enhanced the level even in the control group. These results suggest that the extreme inflammatory responses to endotoxin are prevented in the 40% energy-restricted mice, and corticosterone participates in the preventive effects.

Increases in gastric histidine decarboxylase activity and plasma gastrin level in streptozotocin-induced type 1 diabetic rats.

When type 1 diabetes mellitus was induced in rats by injecting streptozotocin, histidine decarboxylase expression was abnormally up-regulated in a transcriptional level, and 7 d after the injection, the enzyme activity was increased about 3-fold over the control (p < 0.05). When the diabetic rats were administered with insulin for 3 d, the increased histidine decarboxylase activity returned to a normal level in addition to normalization of the plasma glucose level. The plasma gastrin level in the fasting state was also significantly elevated in the diabetic rats, and the insulin treatment normalized the level. In the diabetic rats, fasting gastric acid output increased significantly and gastric pH was lowered. These results suggest that the gastric histidine decarboxylase activity and plasma gastrin level are increased in connection with the depletion of insulin in streptozotocin-induced diabetic rats, and gastric acid secretion is stimulated at a basal level, presumably due to increases in the concentrations of histamine and gastrin in oxyntic mucosa.

Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex.

Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and gamma-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as beta-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that approximately 70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and approximately 75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.

Implanted tumor growth is suppressed and survival is prolonged in sixty percent of food-restricted mice.

To examine the effect of food restriction on immune functions in the tumor-bearing state, mice were divided into a control group (fed 5.0 g diet/d; 71 kJ/d) and a 60% food-restricted group (fed 3.0 g diet/d; 43 kJ/d) at 8-wk of age, and 4 wk later, L1210 tumor cells were inoculated intradermally. In the food-restricted mice, tumor growth was significantly suppressed, and mean survival time after the tumor inoculation was prolonged (P < 0.05). The plasma concentrations of two antitumor cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), were greater in the food-restricted group before tumor inoculation (P < 0. 05). Furthermore, the food-restricted mice had significantly higher plasma levels of IFN-gamma and TNF-alpha after tumor inoculation, although the treatment significantly increased these cytokine levels in both groups. Splenic natural killer cell cytotoxicity was also higher in the tumor-bearing food-restricted mice than in controls (P < 0.05). Food-restricted mice have strong antitumor immunity, and as a result, tumor growth is suppressed and survival time is prolonged in these mice.

Disordered expression of the sucrase-isomaltase complex in the small intestine in Otsuka Long-Evans tokushima fatty rats, a model of non-insulin-dependent diabetes mellitus with insulin resistance.

To clarify the relationship between diabetes mellitus and carbohydrate digestion, the activities of sucrase and isomaltase, which form a complex enzyme (SI complex) on the brush border membranes, were compared in the progression of diabetes mellitus in Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus with insulin resistance, and Long-Evans Tokushima Otsuka (LETO) rats as non-diabetic controls. Until 40 weeks of age, OLETF rats were obese and had a high plasma glucose level, compared to age-matched LETO rats, but the sucrase and isomaltase activities showed no significant differences between the two strains. Oral glucose tolerance test revealed that during 40-48 weeks of age, NIDDM became very severe with advancing insulin resistance in OLETF rats. In OLETF rats, in contrast to LETO rats, at 48 weeks of age, abnormal increases in the sucrase and isomaltase activities occurred, along with a remarkable decrease in body weight and a further great increase in the plasma glucose level in the non-fasting state. Hyperinsulinemia occurred in 20-week-old OLETF rats; however, at 40 and 48 weeks of age, the plasma insulin level in the non-fasting state in OLETF rats was not significantly different from that in LETO rats. The level of mRNA encoding the SI complex increased abnormally in 48-week-old OLETF rats. These results suggest that the advance of insulin resistance leads to an increase in the expression of the SI complex on the transcriptional level.

Isoform selective inhibition and inactivation of human cytochrome P450s by methylenedioxyphenyl compounds.

1. A series of methylenedioxyphenyl compounds were evaluated for their inhibitory and inactivation effects on nine human cytochrome P450 (CYP) activities using microsomes from human B-lymphoblast cells expressing specific human CYP isoforms. 2. Methylenedioxyphenyl compounds which possess a bulky structure such as 1,4-benzothiazine showed substantial inhibition of S-warfarin 7-hydroxylation catalysed by CYP2C9, S-mephenytoin 4'-hydroxylation by CYP2C19, bufuralol 1'-hydroxylation by CYP2D6, and testosterone 6beta-hydroxylation by CYP3A4. Regarding ethoxyresorufin O-deethylation catalysed by CYP1A1 and benzyloxyresorufin O-dealkylation by CYP2B6, the subtle change of a substitution of the 1,4-benzothiazine structure affected the inhibition selectivity. Ethoxyresorufin O-deethylation by CYP1A2, coumarin 7-hydroxylation by CYP2A6, and chlorzoxazone 6-hydroxylation by CYP2E1 were not inhibited by almost any of the methylenedioxyphenyl compounds. The inhibitory effects of methylenedioxyphenyl compounds that possess a short chain amino group on the human CYP isoforms were not significant. 3. The methylenedioxyphenyl compounds inactivated CYP1A1 (k(inact) = 0.034 min(-1) and K(i) = 0.81 microM), CYP2C9 (k(inact) = 0.041 and 0.042 min(-1) and K(i) = 0.56 and 0.15 microM), CYP2D6 (k(inact) = 0.044-0.339 min(-1) and K(i) = 0.21-19.88 microM), and CYP3A4 (k(inact) = 0.076-0.251 min(-1) and K(i) = 0.25-0.69 microM). These results suggested that the methylenedioxyphenyl compounds investigated in this study would be potent mechanism-based inactivators of these human CYP isoforms. In contrast, CYP2B6 and CYP2C19 were not inactivated. 4. The present study suggested that the selectivity of inhibition or inactivation of human CYP isoforms by methylenedioxyphenyl compounds may vary according to the structure of the side chain.

Suppression by water extracts of Sophora plants of sucrose-induced hyperglycemia in rats and inhibition of intestinal disaccharidases in vitro.

Partially purified hot-water extracts of the roots of plants of the Sophora family suppressed the increase in blood glucose concentration of rats in the oral sugar tolerance test. The extracts also inhibited rat intestinal sucrase and maltase. The most potent sample was about 15 times more active than catechin, a positive control, in these experiments.

Low sucrase activity in the small intestine of a senescence-accelerated strain of mouse, SAMP1.

The small intestinal sucrase activity in a senescence-accelerated strain of mouse, SAMP1, was significantly lower than that in other strains, including its control strain, SAMR1. In contrast, the activity of isomaltase, which usually associates with sucrase to form a complex enzyme (SI complex), in SAMP1 was comparable to that in other strains. Thus, the ratio of the sucrase to isomaltase activities (S/I ratio) in SAMP1 was very low (about 0.15), compared with that in other strains (around 0.7). The S/I ratio in SAMP1 was abnormally low, even at a young age, indicating that senescence did not result in the low sucrase activity. Western blot analysis suggests that a large part of the isomaltase subunit occurred alone without the association of the sucrase subunit in this strain. In contrast, Northern blot analysis shows that the level of mRNA for the SI complex in SAMP1 was comparable to that in SAMR1. When the pancreatico-biliary ducts were ligated in SAMP1 to reduce the level of pancreatic proteases, a remarkable increase was observed in the sucrase activity, whereas the isomaltase activity was increased to a much smaller extent. This marked increase in sucrase activity resulted in the S/I ratio increasing to 0.84 18 h after the ligation. These results suggest the sucrase subunit of the SI complex to be abnormally unstable against pancreatic proteases in SAMP1.

The intron 5-inserted form of rat erythropoietin receptor is expressed as a membrane-bound form.

The cDNA encoding an intron 5-inserted form of the erythropoietin receptor (I5Epo-R) has been cloned from rat. DNA sequence analysis reveals that the insertion of intron 5, which consists of 79 bp, causes a shift in reading frame and results in termination in the region of exon 7. The deduced amino acid sequence is composed of 316 amino acid residues, which is a molecular weight of 34220. To study the function of rat I5Epo-R, we established a Chinese hamster ovary cell line expressing rat I5Epo-R. Western blot analysis and binding studies with 125I-recombinant human erythropoietin showed that the transfected cells expressed rat I5Epo-R with a molecular size of 36 kDa as a membrane-bound form, but not as a soluble form, and had a single class of binding sites with a Kd of 700 pM.

Alpha 2-adrenoceptor-mediated antisecretory effect of hypoxia in conscious rats.

Gastric acid secretion is suppressed, resulting in a significant rise in gastric pH, when conscious rats are exposed to hypoxia (Yamaji et al., 1996). When adrenal medullectomized rats were exposed to moderate (10.5% O2) hypoxia for 3 h, gastric acid secretion was restored to nearly the level in normoxia by the adrenal medullectomy. In severe (7.6% O2) hypoxia, the operation also caused an increase in the level of gastric acid output, although the extent was lower than that under 10.5% O2 hypoxic conditions. Gastric pH was normalized by the operation even with 7.6% O2 hypoxia. Similar results were obtained when reserpine, which causes an adrenergic discharge, was administered. When an alpha 2-adrenoceptor blocking agent, yohimbine, was administered, the inhibitory effect of 10.5% and 7.6% O2 hypoxia on gastric acid secretion was almost completely removed. However, neither prazosin (an alpha 1-adrenoceptor blocker) nor propranolol (a beta-adrenoceptor blocker) showed any significant effects on gastric acid output in hypoxia. These results indicate that acute hypoxia stimulated the adrenergic response from the adrenal medulla, and that gastric acid secretion was consequently suppressed through alpha 2-adrenoceptor.

Suppressive effect of insulin on the synthesis of sucrase-isomaltase complex in small intestinal epithelial cells, and abnormal increase in the complex under diabetic conditions.

An abnormally high level of the sucrase-isomaltase (SI) complex in the small intestine of rats with streptozotocin-induced insulin-dependent diabetes mellitus (IDDM) was normalized in 11 h by the administration of insulin, in addition to normalization of the blood glucose level. Phlorizin, an inhibitor of renal glucose reabsorption, also caused normalization of the blood glucose level in the IDDM rats; however, the level of the SI complex was barely changed. When mucosa explants were cultured in a medium, the SI complex synthesized during the cultivation was accumulated as its precursor protein without maturation, owing to the absence of pancreatic proteases, and the amount of the precursor protein that accumulated in the explants was decreased by the addition of insulin into the medium. Further, the mRNA level of the SI complex in the explants incubated with insulin was obviously lower than that in the absence of insulin. These results indicate that insulin has a suppressive effect on the synthesis of the SI complex, presumably by decreasing the transcriptional level of the gene encoding the complex, in small-intestinal epithelial cells. Thus the synthesis of the SI complex might exceed normal levels in the epithelial cells as a direct result of the depletion of insulin under IDDM conditions.

Inositol 1,4,5-trisphosphate and cyclic ADP-ribose mobilize Ca2+ in a protist, Euglena gracilis.

Inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) released Ca2+ from microsome fraction prepared from Euglena gracilis in dose-dependent manners. Caffeine, which also induced Ca2+ release from the microsomes, caused desensitization of the Ca2+ response to cADPR, although the Ca2+ response to InsP3 was not affected by caffeine. Further, ruthenium red inhibited the Ca2+ release induced by cADPR, but not by InsP3. These results suggest that cADPR functions as an endogenous messenger to activate a caffeine-sensitive, Ca(2+)-release mechanism, whereas InsP3 induces Ca2+ release by a distinct mechanism in E. gracilis.

Mitochondrial NADH- or NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes.

Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.

Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA.

To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with 125I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (Kd = 860 pM) and that the number of sites/cell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endothelial cells as a competence factor.

Hypoxia inhibits gastric emptying and gastric acid secretion in conscious rats.

This study examines the effects of hypoxia in the gastric function in conscious rats which adapted to a meal-feeding schedule, that allowed free access to a high protein (HP) diet (550 g casein/kg diet, Exp.1,2 and 4), a normal protein (NP) diet (200 g casein/kg diet, Exp.3) or a nonpurified rat (NPR) diet (Exp. 5 and 6) for 4 h every day for 2 wk. In Exp. 1, after 4 h of consuming the HP diet, rats were exposed to 7.6 or 10.5% O2 normobaric hypoxia. Hypoxia delayed the excretion of urinary urea for 12 h. In Exp.2 and 3, when rats were exposed to 7.6%O2 after 4 h of consuming the HP diet and exposed to 10.5% O2 after 4 h of consuming the NP diet, respectively, a significant delay in gastric emptying was found in the hypoxic rats. In Exp. 4, when rats were exposed to 7.6 O2 hypoxia after 4 hr of eating the HP diet, the plasma gastrin concentration in the 7.6% O2 hypoxic rats was 2.3-fold that of the normoxic rats after 6 h of hypoxia. Furthermore, when rats that did not consume any HP diet on the day of the experiment were exposed to 7.6 or 10.5% O2 hypoxia, the plasma gastrin concentration was higher in both hypoxic groups than in the normoxic group after 3 and 6 of hypoxia. In Exp. 5, rats that were not fed the NPR diet on the day of study were exposed to 7.6 or 10.5% O2 hypoxia for 3 h after pylorus ligation. Hypoxia inhibited the secretion of gastric acid and elevated the plasma gastrin concentration. In Exp. 6, unfed rats that had been consuming the NPR diet were exposed to 7.6% O2 hypoxia for 3 h after pylorus ligation and were orally administered HCl. The rise of the gastrin concentration due to hypoxia was completely inhibited by oral HCl. These results demonstrate that hypoxia inhibits gastric emptying and gastric acid secretion and that the inhibitory effect of hypoxia on gastric acid secretion stimulates gastrin release through positive feedback regulation.

Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis.

Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin.

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.