Influence of alloxan-induced diabetes and selenite treatment on blood glucose and glutathione levels in mice

Many clinical studies reported that diabetic patients had lower glutathione contents in erythrocytes or plasma. Recently, selenium, an essential trace element with well-known antioxidant characteristics, has been found to have insulin-mimetic properties. But seldom information is available about the influence of selenium on glutathione changes induced by diabetes mellitus in animals. Therefore, this study was designed to compare the impacts of selenite treatment on glutathione (GSH) levels of blood and tissues such as brain, kidney, liver, spleen and testis in mice. Four groups were used in this study: a control group, a diabetic group, a selenite-treated normal group and a selenite-treated diabetic group. Selenite was administered to the mice for 4 weeks with an oral dose of 2 mg kg(-1) day(-1) by gavage. The blood glucose level, and GSH level in blood and tissues were determined. The results show that the selenite-treated diabetic group had significantly lower blood glucose levels than the diabetic group. Moreover, alloxan-induced diabetes significantly decreased GSH levels in blood, kidney, liver and testis compared to the controls. Selenite treatment of the diabetic mice only improved the GSH levels in liver and brain. On the other hand, selenite administered to the normal mice reduced GSH levels in the liver compared to the controls. In conclusion, this study suggests that selenite treatment of diabetic mice with an effective dose would be beneficial for the antioxidant system of liver and brain although it exerts a toxic effect on the liver of normal mice.     

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.     

肥胖犬白细胞中相关基因及血浆生化指标的变化

目的观察不同肥胖时期的犬血浆中相关生化指标和外周血液白细胞(peripheral blood leukocytes,PBL)中胰岛素和脂联素通路、能量和脂类代谢相关基因的变化,为开发肥胖犬代谢紊乱的早期诊断法提供依据。方法选用到动物医院进行常规体检的不同品种的犬59只,按照5分制的身体评分指数分为正常犬(26只)、超重犬(28只)和肥胖症犬(5只),分别检测血浆中生化代谢指标(葡萄糖、血尿素氮、肌氨酸酐、总胆固醇、总蛋白质、三酸甘油酯、乳酸脱氢酶、碱性磷酸酶、天冬氨酸转氨酶、丙氨酸转氨酶、非酯化脂肪酸、脂联素和免疫反应胰岛素)和PBL中相关基因[胰岛素受体底物(IRS-1,-2)、磷脂酰肌醇-3-激酶(PI3-K p85α),苹果酸脱氢酶(malate de-hydrogenase,MDH)、葡萄糖-6-磷酸脱氢酶(G6DPH)、脂肪酸合酶(FAS)、脂联素受体(ADIPOR1,2)]mRNA表达量。结果与对照犬相比,超重犬和肥胖犬血浆中的胰岛素含量显著升高,肥胖犬非酯化脂肪酸、总胆固醇和三酸甘油酯显著升高。肥胖犬PBL中胰岛素下游相关基因IRS-2、脂类代谢基因FAS和能量代谢基因MDH的mRNA表达量显著下降。结论肥胖犬患有高胰岛素血症和严重的脂类代谢紊乱,胰岛素下游基因和其他代谢相关基因的显著下降可能提示肥胖犬患有胰岛素抵抗和能量代谢紊乱。     

Quantification of hepatic carbohydrate metabolism in conscious mice using serial blood and urine spots

In vivo studies of hepatic carbohydrate metabolism in (genetically modified) conscious mice are hampered by limitations of blood and urine sample sizes. We developed and validated methods to quantify stable isotope dilution and incorporation in small blood and urine samples spotted onto filter paper. Blood glucose and urinary paracetamol-glucuronic acid were extracted from filter paper spots reproducibly and with high yield. Fractional isotopomer distributions of glucose and paracetamol-glucuronic acid when extracted from filter paper spots were almost identical to those isolated from the original body fluids. Rates of infusion of labeled compounds could be adjusted without perturbing hepatic glucose metabolism. This approach was used in mice to find the optimal metabolic condition for the study of hepatic carbohydrate metabolism. In fed mice, no isotopic steady state was observed during a 6-h label-infusion experiment. In 9-h-fasted mice, isotopic steady state was reached after 3 h of label infusion and important parameters in hepatic glucose metabolism could be calculated. The rate of de novo glucose-6-phosphate synthesis was 143 +/- 17 micromol kg(-1) min(-1) and partitioning to plasma glucose was 79.0 +/- 5.2%. In 24-h-fasted mice, abrupt changes were noticed in whole body and in hepatic glucose metabolism at the end of the experiment.     

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species

Previous work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1 % of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi.     

Gene expression profile in bone of diabetes-prone BB/OK rats fed a high-fat diet

A high-fat diet (HFD) has been recognized as a risk factor for diseases such as dyslipidemia, atherosclerosis, obesity, and osteoporosis. However, studies analyzing gene expression after HFD in bone are rare. That prompted us to analyze the expression of selected genes in bone of 4-week-old diabetes-prone B(io)B(reeding) rats. Two breeding pairs were fed a HFD (+10 % tallow) or were fed a normal diet (ND; Ssniff R-Z) before mating and afterward during pregnancy. After the birth of progeny, parents continued to be given HFD or ND until the progeny was weaned (3 weeks). Thereafter, offspring were weaned and were fed the same food as their parents up to an age of 4 weeks. Body weight was measured at an age of 4 weeks, and subsequently 13 HFD rats and 13 ND rats were killed and the tibial bone was harvested to analyze the expression of 53 genes in bone. All rats fed HFD were significantly heavier than rats fed ND after 3 and 4 weeks. The diet also influenced the expression of genes in bone. There were significant differences in 20 out of 53 genes studied between rats fed HFD compared with rats fed ND. Four out of 20 had a lower and 17 out of 20 genes a higher expression in HFD rats, but differences in gene expression showed obvious differences between males and females. There were only two genes that were similarly different between males and females: Bmp4 and Atf4. Two genes, Foxg1 and Npy, were inversely expressed in males and females. It seems that the gene expression is differently regulated by diet during pregnancy and later in life between males and females. Nevertheless, it cannot be excluded that HFD also acts as an epigenetic factor in the development of offspring in utero.     

Relative argininosuccinate synthetase mRNA levels and gene copy number in canavanine-resistant lymphoblasts

Mutants resistant to the arginine analogue, canavanine, have been isolated from two normal lymphoblast lines, MGL8B2 and MGL33. These mutants constitutively express up to 200-fold higher amounts of structurally normal argininosuccinate synthetase, the urea cycle enzyme that converts citrulline to argininosuccinate. Relative levels of argininosuccinate synthetase mRNA were compared among normal and canavanine-resistant lines using in vitro translation of poly(adenylic acid) RNA and blot hybridization of total cytoplasmic RNA to an argininosuccinate synthetase cDNA. Both of these approaches indicated that the canavanine-resistant lines contain increased steady-state levels of synthetase-specifc mRNA relative to their sensitive parents and that these were roughly correlated with levels of enzyme activity. Blot hybridization of Eco RI-digested genomic DNA preparations revealed no detectable differences in argininosuccinate synthetase structural gene copy number between normal and canavanine-resistant lymphoblasts, demonstrating that the canavanine-resistant phenotype is not caused by gene amplification.     

C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output

Adipose tissue-derived adipokines play important roles in controlling systemic insulin sensitivity and energy balance. Our recent efforts to identify novel metabolic mediators produced by adipose tissue have led to the discovery of a highly conserved family of secreted proteins, designated as C1q/TNF-related proteins 1-10 (CTRP1 to -10). However, physiological functions regulated by CTRPs are largely unknown. Here we provide the first in vivo functional characterization of CTRP3. We show that circulating levels of CTRP3 are inversely correlated with leptin levels; CTRP3 increases with fasting, decreases in diet-induced obese mice with high leptin levels, and increases in leptin-deficient ob/ob mice. A modest 3-fold elevation of plasma CTRP3 levels by recombinant protein administration is sufficient to lower glucose levels in normal and insulin-resistant ob/ob mice, without altering insulin or adiponectin levels. The glucose-lowering effect in mice is linked to activation of the Akt signaling pathway in liver and a marked suppression of hepatic gluconeogenic gene expression. Consistent with its effects in mice, CTRP3 acts directly and independently of insulin to regulate gluconeogenesis in cultured hepatocytes. In humans, alternative splicing generates two circulating CTRP3 isoforms differing in size and glycosylation pattern. The two human proteins form hetero-oligomers, an association that does not require interdisulfide bond formation and appears to protect the longer isoform from proteolytic cleavage. Recombinant human CTRP3 also reduces glucose output in hepatocytes by suppressing gluconeogenic enzyme expression. This study provides the first functional evidence linking CTRP3 to hepatic glucose metabolism and establishes CTRP3 as a novel adipokine.     

Novel SIRT1 activator MHY2233 improves glucose tolerance and reduces hepatic lipid accumulation in db/db mice

The NAD⁺-dependent deacetylase SIRT1, which is associated with the improvement of metabolic syndromes, such as type 2 diabetes, is a well-known longevity-related gene. Several in vitro and in vivo studies have shown the known protective effects of SIRT1 activators, such as resveratrol and SRT1720, on diabetes- or obesity-induced fatty liver and insulin resistance. Here, we newly synthesized 18 benzoxazole hydrochloride derivatives based on the structure of resveratrol and SRT1720. We performed an in vitro SIRT1 activity assay to identify the strongest SIRT1 activator. The assay confirmed MHY2233 to be the strongest SIRT1 activator (1.5-fold more potent than resveratrol), and docking simulation showed that the binding affinity of MHY2233 was higher than that of resveratrol and SRT1720. To investigate its beneficial effects, db/db mice were orally administered MHY2233 for 1?month, and various metabolic parameters were assessed in the serum and liver tissues. MHY2233 markedly ameliorated insulin signaling without affecting body weight in db/db mice. In particular, the mRNA expression of lipogenic genes, such as acetyl CoA carboxylase, fatty acid synthase, and sterol regulatory element-binding protein, which increased in db/db mice, decreased following oral treatment with MHY2233.In conclusion, the novel SIRT1 activator MHY2233 reduced lipid accumulation and improved insulin resistance. This finding may contribute toward therapeutic approaches for fatty liver disease and glucose tolerance.     

mRNA levels of SREBP-1c do not coincide with the changes in adipose lipogenic gene expression

Physiological differences in lipid metabolism exist according to adipose sites. To delineate at which step such gene regulation could occur, mRNA levels of various proteins involved in the overall lipogenic process were determined in subcutaneous (SC) and retroperitoneal (RP) adipose tissues. Fatty acid synthase, malic enzyme, ATP citrate lyase, insulin-sensitive glucose transporter, and glucose-6-phosphate dehydrogenase mRNA levels were coordinately reduced (by up to 50-fold) during fasting in RP and in SC relative to fed rats, and restored or overexpressed (by up to 5- to 6-fold) during refeeding. The response was most often delayed and lower in SC compared to RP. This could contribute to site-specific differences. Interestingly, SREBP-1c mRNA levels were markedly decreased by fasting in SC but remained unchanged in RP. Refeeding tended to restore levels close to fed group values. We conclude that mRNA levels of SREBP-1c do not coincide with the expected changes in adipose lipogenic gene expression of fasted/refed rats.     

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of (13)C-labelled lipids obtained from gonococci grown in a synthetic medium with (13)C-labelled lactate and unlabelled glucose (culture A), (13)C-labelled glucose alone (culture B) or (13)C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of (13)C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, (14)C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [(14)C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [(14)C]lactate was used in culture A, than when [(14)C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.     

Competition between Salvinia minima and Spirodela polyrhiza mediated by nutrient levels and herbivory

We investigated the effects of initial biomass, nutrients, herbivory, and competition with Spirodela polyrhiza (L.) Schleid on Salvinia minima Baker biomass and density. S. minima populations were subjected to two levels of herbivory (control vs. two adults per plant) from the weevil Cyrtobagous salviniae Calder and Sands and eight levels of competition from S. polyrhiza, while growing in high (5 mg N l⁻¹) or low (0.5 mg N l⁻¹) nutrient conditions. Herbivory was the most important factor in S. minima biomass production while competition or fertility had no measurable impact. In contrast, S. polyrhiza biomass was mediated primarily by nutrients, not competition. There was no herbivory treatment for this plant. S. polyrhiza was superior to S. minima at converting nutrients to biomass but this did not give it a competitive advantage since S. minima biomass was unchanged when herbivory was absent. S. minima can generally overtop S. polyrhiza which, in turn, can form multiple layers within its mat. These characteristics may act to lessen competition between these species, thereby permitting their habitat sharing.