14C-glucose Oxidation Research Articles

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation, protein synthesis and protein breakdown in mouse C2C12 myotubes

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor 14C-glucose oxidation or to measure protein synthesis by incorporation of 3H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P<0.01, n=3 trials), whereas bovine insulin (1 µM) stimulated glucose oxidation (P<0.05, n=3 trials). Treatment with AGP in combination with insulin reduced 14C-glucose oxidation (P<0.05, n=3 trials), similar to the effect of AGP alone. Glucose transport, as measured by 3H-deoxyglucose uptake, was increased by 38% with 1 µM insulin (P<0.05, n=3 trials), whereas AGP alone increased glucose uptake by 36% (P<0.05, n=3 trials). The combination of insulin and AGP in the medium resulted in an 88% increase in glucose uptake (P<0.01, n=3 trials). Protein synthesis was measured by 3H-tyrosine incorporation into C2C12 myotubes. Insulin stimulated a 18% increase in 3H-tyrosine incorporation (P<0.05, n=6 trials). The incorporation of 3H-tyrosine into myotubes was reduced by 20% with AGP incubation (P<0.01, n=6 trials), like the 20% decrease in 3H-tyrosine incorporation in response to the combination of AGP and insulin (P<0.01, n=6 trials). Protein breakdown, as measured by the release of 3H-tyrosine from C2C12 myotubes, was reduced 27% by insulin (P<0.01, n=6 trials). Treatment with AGP had no effect on protein breakdown (P>0.05, n=6 trials), whereas incubation with both AGP and insulin reduced 3H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.

Transient gestational exposure to drinking water containing excess hexavalent chromium modifies insulin signaling in liver and skeletal muscle of rat progeny

Chromium (Cr), an essential micronutrient potentiates insulin action, whereas excess hexavalent Cr (CrVI) acts as an endocrine disruptor. Pregnant mothers living in areas abutting industries using the metal and chromite ore dumps are exposed to ground water contaminated with Cr. Nevertheless, the impact of prenatal exposure to excess CrVI on insulin signaling in the progeny remains obscure. We tested the hypothesis “transient gestational exposure to drinking water containing excess CrVI may modify insulin signaling during postnatal life”. Pregnant Wistar rats were given drinking water containing 50, 100 and 200 ppm CrVI (K2Cr2O7) from gestational day 9–14 encompassing the period of organogenesis; the male progenies were tested at postnatal day 60. Neither fasting blood glucose nor oral glucose tolerance was altered in CrVI treated progeny. Nevertheless, western blot detection pointed out attenuated expression level of insulin receptor (IR), its downstream signaling molecules (IRS-1, pIRS-1Tyr632, Akt and pAktSer473) and organ specific glucose transporters (GLUT2 in liver and GLUT4 in gastrocnemius muscle), along with a significant increase in serum insulin level in male progenies exposed to CrVI. While 14C-2-deoxy glucose uptake increased in the liver, the same decreased in the skeletal muscle whereas, 14C-glucose oxidation recorded a consistent decrease in both tissues of CrVI exposed rats. These findings support our hypothesis and suggest that transient gestational exposure to excess CrVI may affect insulin signaling and glucose oxidation in the progeny, predictably rendering them vulnerable to insulin resistance.

Effects of DEHP and its metabolite MEHP on insulin signalling and proteins involved in GLUT4 translocation in cultured L6 myotubes.

Di-(2-ethyl hexyl) phthalate (DEHP) is the plasticizer used in variety of medical and consumer products to impart structural flexibility. DEHP and its primary metabolite mono-(2-ethyl hexyl)phthalate (MEHP) posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. Experimental and epidemiological data have shown that DEHP affects blood glucose homeostasis. However, direct effect of DEHP and its metabolite MEHP on insulin signal transduction and glucose transporter 4 (GLUT4) translocation remain obscure. The present study was delineated to decipher the direct effects of DEHP and MEHP on insulin signal transduction and proteins involved in GLUT4 translocation in cultured L6 myotubes, the rat skeletal muscle model. For this study we have exposed cells with 50 and 100μM DEHP and MEHP for 24h followed by insulin stimulation for 20min. GLUT4 level in both cytosol and plasma membrane fractions were analysed by western blot analysis and found to be significantly decreased. Further, DEHP and MEHP treatment significantly altered the insulin signalling molecules and proteins involved in GLUT4 translocation (Rab8A (Ras related proteins in skeletal muscle), insulin - regulated amino peptidase (IRAP), synaptosomal - associated protein 23 (SNAP23), Syntaxin4, Munc18c) from cytosol to plasma membrane. Impaired GLUT4 in the plasma membrane resulted in decreased 14C-deoxy glucose uptake. 14C-glucose oxidation and glycogen content were also significantly decreased in treated groups. In essence, the present study is first of its kind to show the direct adverse effects of DEHP and MEHP on insulin signal transduction and GLUT4 translocation in cultured L6 myotubes. Further, MEHP is found to be more effective than DEHP as a result of its differential structure and physico-chemical properties.

Glucose Transporter-4 Facilitates Insulin-Stimulated Glucose Uptake in Osteoblasts.

Recent studies have identified the osteoblast as an insulin responsive cell that participates in global energy homeostasis. Here, we show that glucose transporter-4 (Glut4) is required for insulin-dependent uptake and oxidation of glucose in mature osteoblasts. In primary cultures of mouse osteoblasts, insulin increased uptake and oxidation of 14C-glucose in a dose-dependent fashion but did not significantly affect uptake or oxidation of 14C-oleate. In vitro, undifferentiated osteoblasts expressed 3 high-affinity Gluts: Glut1, Glut4, and Glut3. However, although levels of Glut1 and Glut3 remained constant during the course of osteoblast differentiation, Glut4 expression increased by 5-fold in association with enhanced insulin-stimulated glucose uptake. Glut4 ablation in osteoblasts in vitro eliminated insulin-stimulated glucose uptake, reduced proliferation and diminished measures of osteoblast maturation. In vivo, Glut4 expression was observed in osteoblasts, osteocytes, and chondrocytes at a level approaching that observed in adjacent skeletal muscle. To determine the importance of Glut4 in bone in vivo, we generated mice lacking Glut4 in osteoblasts and osteocytes (ΔGlut4). ΔGlut4 mice exhibited normal bone architecture but exhibited an increase in peripheral fat in association with hyperinsulinemia, β-cell islet hypertrophy, and reduced insulin sensitivity. Surprisingly, the expression of insulin target genes in liver, muscle, and adipose from ΔGlut4 mice were unchanged or increased, indicating that alterations in glucose homeostasis were the result of reduced clearance by bone. These findings suggest that Glut4 mediates insulin-stimulated glucose uptake by mature osteoblasts/osteocytes and that the magnitude of glucose use by bone cells is sufficient to impact global glucose disposal in the mouse.

β-cell regenerative efficacy of a polysaccharide isolated from methanolic extract of Tinospora cordifolia stem on streptozotocin -induced diabetic Wistar rats

The use of herbal supplements either as extracts or plant-derived individual molecules has significantly increased in the process of drug discovery and development for their potential efficacy or reduced risk in treating human disorders. Tinospora cordifolia (T. cordifolia) is a widely used herbal source to treat various human ailments, including diabetes mellitus. The present study was aimed on evaluating the antidiabetic property of a novel polysaccharide isolated from the methanolic extract of T. cordifolia stem. Bioassay guided fractionation was followed to isolate a compound from the methanol extract. The compound was administered orally at a dose of 20 mg/kg.b.wt for 60 days to control and STZ-induced diabetic male Wistar rats. It was found that plasma glucose was significantly (p < 0.05) reduced compared to normal. Oral administration of the compound significantly decreased HBA1c, triglycerides and total cholesterol and at the same time markedly increased hemoglobin, tissue glycogen and HDL cholesterol. Also the compounds restored the altered carbohydrate metabolizing enzymes, insulin, C-peptide, 14C-glucose oxidation levels to near normal. In addition, the histological studies revealed that there was regeneration of β-cells in the pancreatic sections. The expression of Glut-4 mRNA and protein in the gasrtocnemius muscle were significantly enhanced after the compound treatment. These results confirm that the novel polysaccharide possesses hypoglycemic, glucose oxidizing, hypolipidemic and β-cell regenerative properties and hence it could be developed into potential oral hypoglycemic drug with lesser side effects.

Evaluation of antidiabetic property of Andrographis paniculata powder in high fat and sucrose-induced type-2 diabetic adult male rat

ObjectiveTo evaluate the antidiabetic effect of the aerial part of Andrographis paniculata (A. paniculata) powder (500 mg/kg body weight) in high fat and sucrose-induced type-2 diabetic rat model. MethodsThe fasting blood glucose, oral glucose tolerance test, serum insulin, lipid profile, mRNA and protein levels of insulin signaling molecules, 14C-2 deoxy glucose uptake and 14C glucose oxidation in liver were checked. ResultsIn the type-2 diabetes-induced group, the fasting blood glucose, oral glucose tolerance, serum insulin, lipid profile, glucose uptake and oxidation, Akt and glucose transporter 2 mRNA, insulin receptor and glucose transporter 2 protein (both cytosolic and plasma membrane) and phosphorylation of insulin receptor substrate 1 and Akt were impaired. A. paniculata was able to successfully reinstate this impairment. In addition to this, A. paniculata did not cause a hypoglycemic condition in normal rat, affirming its activity in hyperglycemic state alone. ConclusionsA. paniculata possesses significant antidiabetic and antihyperlipidemic activities in high fat and sucrose-induced type-2 diabetic rat and the molecular actions at the level of insulin signaling molecules in liver reinforce it.

Effect of bisphenol-A on insulin signal transduction and glucose oxidation in skeletal muscle of adult male albino rat

The estrogenic monomer bisphenol-A (BPA) is an endocrine-disrupting chemical used in the production of epoxy resins, plastic food and beverage containers, leading to ubiquitous human exposure. Environmentally relevant doses of BPA have profound effects on mice endocrine pancreas. It increases pancreatic insulin content and favors postprandial hyperinsulinemia and insulin resistance in male mice. Skeletal muscle plays a crucial role in maintaining systemic glucose metabolism. In the present study, we investigated the possible effects of BPA on insulin-signaling molecules and glucose oxidation in skeletal muscle of male rat. Adult male Wistar albino rats were divided into three groups. Group I: control (vehicle treated) and groups II and III were administered with BPA orally (20 and 200 mg/kg bw/day, respectively). Although there was no change in the levels of insulin receptor (IR), Akt (protein kinase B) and glucose transporter-4 (GLUT4) messenger RNA, BPA significantly decreased the IR, Akt and GLUT4 protein levels (both plasma membrane and cytosolic fraction) of the gastrocnemius muscle. There was an increase in serum insulin and decrease in serum testosterone levels but fasting blood glucose level remained unaltered. In conclusion, BPA has adverse effects on phosphorylation of Akt, GLUT4 translocation and (14)C-glucose oxidation.

Methyl-β-cyclodextrin alters adipokine gene expression and glucose metabolism in swine adipose tissue

This study was designed to determine whether methyl-β-cyclodextrin (MCD) can substitute for albumin in incubation medium for neonatal swine adipose tissue explants, or whether MCD affects metabolism and cytokine expression. Subcutaneous adipose tissue explants (100 ± 10 mg) were prepared from 21-day-old pigs. Explants were incubated in medium 199 supplemented with 25 mM HEPES, 1.0 nM insulin at 37°C. The medium also contained bovine serum albumin (BSA) or MCD at 0%, 0.05%, 0.1%, 0.2% or 0.3%. Tissue explants were treated with these media for 1 h and then switched to the same basal incubation medium containing 0.05% BSA. Explants were removed from basal medium at 2 or 8 h of incubation, and real-time PCR was performed to assess expression of tumor necrosis α (TNF) and interleukin 6 (IL6), acetyl CoA carboxylase (ACAC) and fatty acid synthase (FASN). Alternatively, rates of 14C-glucose oxidation and lipogenesis were monitored ± insulin (100 nM), following MCD treatment. Incubation with BSA had minimal effects on gene expression or adipose tissue metabolism, only producing a doubling in TNF mRNA abundance (P < 0.01). Treatment with MCD increased TNF mRNA abundance by eightfold (P < 0.009), whereas IL6 gene expression increased a 100-fold (P < 0.001) with a suppression in ACAC and FASN expression (P < 0.01). This was paralleled by MCD inhibition of insulin-stimulated glucose oxidation and lipogenesis (P < 0.001). Addition of a TNF antibody to the incubation medium alleviated this inhibition of insulin-stimulated glucose metabolism by ∼30% (P < 0.05).

Effects of dehydroepiandrosterone (DHEA) and lactate on glucose uptake in the central nervous system

Dehydroepiandrosterone (DHEA) prevents brain aging, enhances the cerebral metabolism and interacts with energy substrates. The interaction between lactate and DHEA on glucose uptake and lactate oxidation by various nervous structures was investigated and results demonstrate that the 2- 14C-deoxiglucose (2- 14C-Dglucose) uptake was stimulated by 10 mM lactate in the hypothalamus and olfactory bulb, inhibited in the cerebral cortex and cerebellum, and unaffected in the hippocampus. We also show that, in both the cerebral cortex and hypothalamus, 14C-lactate oxidation was higher than 14C-glucose oxidation ( p ≤ 0.001), demonstrating a relevant role for lactate as energy substrate. The interaction of lactate and 10 −8 M DHEA was tested and, although DHEA had no significant effect on uptake in the cerebellum, hippocampus, or hypothalamus, 10 −8 M DHEA increased the 2- 14C-Dglucose uptake in the cerebral cortex in the presence of lactate ( p ≤ 0.001), and in the olfactory bulb in the absence of lactate ( p < 0.05). However, DHEA had no significant effect on 14C-lactate oxidation. We suggest that DHEA improves glucose uptake in specific conditions. Thus, DHEA may affect CNS metabolism and interact with lactate, which is the most important neuronal energy substrate, on glucose uptake.

Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg(-1) of body weight·day(-1), 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial (14)C-glucose and (14)C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

Insulin mimetic impact of Catechin isolated from Cassia fistula on the glucose oxidation and molecular mechanisms of glucose uptake on Streptozotocin-induced diabetic Wistar rats

Diabetes mellitus is the most common and serious metabolic disorder among people all over the world. Many plants have successfully been used to overcome this problem. Cassia fistula, an ethnomedicnal plant, is widely used in Indian medicine to treat diabetes. Methanol extract of stem of plant, reduced the blood glucose levels in Streptozotocin-induced diabetic rats. Bioassay guided fractionation was followed to isolate Catechin from methanol extract. Catechin was administered to Streptozotocin (60mg/kg b.w.)-induced diabetic male Wistar rats at different doses (5, 10, 20mg/kg b.w.) for 6 weeks to assess its effect on fasting plasma glucose. The plasma glucose was significantly (p<0.05) reduced when compared to the control. Oral administration of Catechin (20mg/kg b.w.) markedly increased tissue glycogen, and (14)C-glucose oxidation without any change in plasma insulin and C-peptide. Catechin restored the altered Glucokinase, glucose-6 Phosphatase, Glycogen Synthase and Glycogen Phosphorylase levels to near normal. GLUT4 mRNA and protein expression were enhanced after Catechin treatment. The results of this experimental study indicated that Catechin possesses hypo-glycemic, Glucose oxidizing and insulin mimetic activities and hence it could be used as a drug for treating diabetes.

The beneficial effect of fructose and glucose on in vitro maturation and the fertilization of porcine oocytes.

This study was conducted to investigate the effects of supplementing fructose in the culture medium on in vitro maturation (IVM), in vitro fertilization (IVF), and metabolism of porcine oocytes. Porcine oocytes were matured in vitro in modified North Carolina State University 37 medium (NCSU-37) and then supplemented with either glucose (5.5 mM), fructose (5.5 mM), or glucose (2.75 mM) plus fructose (2.75 mM). The maturation and fertilization of oocytes, and the incorporation and oxidation of 14C-glucose, 14C-fructose, and 14C-methionine in oocytes at different stages of development were examined. The supplementation of glucose plus fructose significantly promoted (P < 0.05) oocytes germinal vesicle break down (GVBD), maturation to metaphase II (MII), penetration by spermatozoa, and male pronuclear formation compared with glucose. The incorporation and oxidation of 14C-methionine into the oocyte significantly increased (P < 0.05) with glucose plus fructose supplementation than glucose. A significantly higher (P < 0.05) rate of incorporation and oxidation was achieved with 14C-fructose compared to 14C-glucose. Glucose plus fructose supplementation improved maturation, penetration by spermatozoa, male pronuclear formation, and energy metabolism by porcine oocytes.

Sex steroids enhance insulin receptors and glucose oxidation in Chang liver cells

Background The present study was designed to assess the effect of sex steroids (testosterone and 17β-estradiol) on insulin receptor expression, insulin binding and glucose oxidation in human liver cell line. Methods Non-malignant Chang liver cells were treated with different concentrations of testosterone and 17β-estradiol dissolved in serum free medium for 24 h to identify the effective dose of both steroids for further studies. Cells with 70–80% confluency were challenged with testosterone (0.1 µmol/l), 17β-estradiol (0.1 µmol/l) and their combination along with insulin as a positive control for 24 h. After the treatment period, insulin receptor mRNA expression, cell surface insulin binding and 14C-glucose oxidation were assessed. Results Both testosterone and 17β-estradiol significantly increased the insulin receptor mRNA expression, cell surface insulin binding and 14C-glucose oxidation compared to basal, but the increase was not at par with the effect of insulin. Compared to individual effects of testosterone and 17β-estradiol, their combination significantly increased the glucose oxidation similar to that of insulin. Conclusion It is concluded from the present study that testosterone and 17β-estradiol can directly enhance insulin receptor mRNA expression, insulin binding and glucose oxidation in Chang liver cells and thereby glucose metabolism.

Effects of corticosterone deficiency and its replacement on leydig cell steroidogenesis

Clinical and experimental studies have shown the adverse effects of glucocorticoid deficiency/metyrapone treatment on testicular Leydig cell testosterone production. However, molecular mechanisms that underlie the effects of glucocorticoid deficiency on Leydig cell steroidogenesis are not yet determined. Therefore, the present study was designed to assess the mechanism of this phenomenon. Following metyrapone-induced corticosterone deficiency, serum testosterone, and Leydig cell 14C-glucose oxidation were decreased. StAR mRNA and protein levels were significantly increased in Leydig cells of corticosterone-deficient animals. mRNA levels and the specific activities of P(450)scc and 17beta-HSD were decreased by corticosterone deficiency, whereas the activity and mRNA of 3beta-HSD were increased. Simultaneous administration of corticosterone prevented its deficiency-induced changes in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell testosterone production by decreasing the activities of steroidogenic enzymes and their mRNA expression and glucose oxidation.

Assessment of in vitro effects of metyrapone on Leydig cell steroidogenesis

Metyrapone, a specific inhibitor of 11β-hydroxylase inhibits glucocorticoid production and it is used in the diagnosis/treatment of hypercortisolism and also to test the functional integrity of hypothalamo-pituitary-adrenal axis. To assess the impact of glucocorticoid deficiency, this drug is preferred over adrenalectomy, which eliminates all the hormonal secretions of the adrenal cortex and medulla. However, whether metyrapone has any direct effect on the extra-adrenocortical cellular or tissue functions remains to be resolved. Our previous study showed a depressed testicular Leydig cell testosterone production in rats treated with metyrapone. Therefore, the present study was designed to examine the possible direct effect of metyrapone on testicular Leydig cell steroidogenesis in vitro. Leydig cell viability and the reactive oxygen species (ROS) concentration were not altered by any of the concentration of metyrapone tested. The efficacy of Leydig cell testosterone production under basal as well as LH-stimulated condition was not altered by metyrapone treatment. Further, Leydig cellular 14C-glucose oxidation, the activity and mRNA levels of cytochrome side chain cleavage (P 450scc), 3β- and 17β-hydroxysteroid dehydrogenase (3β-HSD and 17β-HSD) were not altered in metyrapone-treated cells. Therefore, it is concluded from the present study that metyrapone has no direct effect on Leydig cell testosterone production and, therefore, changes recorded in the in vivo studies are exclusively due to corticosterone deficiency.

Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa.

Aim: The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin-V (BSA-V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 6h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA-V), fatty acid free BSA (BSA-FAF), polyvinyl alcohol+ main fatty acids bound to BSA-V (PVA+FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of 14C-glucose were evaluated during 6h of incubation. Results: The results show that the BSA-V was superior to BSA-FAF and PVA in improving motility and AR. Viability was significantly increased (P<0.05) by only BSA-V compared with PVA. When the main fatty acids compound of BSA-V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA-V. The rate of incorporation and oxidation of 14C-glucose were significantly increased (P<0.05) by BSA-V compared with BSA-FAF and PVA. Fatty acids bound to BSA-V are important for improvement of sperm functions. Conclusions: The present study postulates that fatty acids bound to BSA-V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and 14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 micromol/L fluorocitrate. However, 20 and 100 micromol/L fluorocitrate reduced lactate oxidation by 55 +/- 20% and 68 +/- 12%, respectively (p < 0.05 for both). Twenty and 100 micromol/L fluorocitrate reduced 14C-glucose oxidation by 50 +/- 14% (p < 0.05) and 24 +/- 19% (ns), respectively. Addition of non-radioactive lactate to (14)C-glucose plus fluorocitrate decreased 14C-glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa.

Background and Aims: Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa. Methods: Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4h in modified Beltsville thawing solution supplemented with 0, 20 or 40ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of 14C-glucose were evaluated during 0-4h of incubation. Results: The results show that the supplementation of relaxin (especially at 20ng/mL) in the thawing solution improved sperm motility significantly (P<0.05) at 1-3h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly (P<0.05) when the spermatozoa were treated with 20ng/mL relaxin. Viability was not significantly (P>0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of 14C-glucose were increased in correlation with AR up to 4h of incubation. Conclusion: We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5: 215-220).

Effects of excess corticosterone on NADPH generating enzymes and glucose oxidation in Leydig cells of adult rats

Clinical and experimental studies have shown the adverse effects of excess glucocorticoid on testicular testosterone production. The NADPH co-enzyme has been recognized as an important factor that regulates several steps in steroidogenesis, while glucose oxidation acts as a limiting factor on testicular testosterone production. Nevertheless, the impact of excess corticosterone, the stress hormone on testicular NADPH availability and glucose oxidation is unknown. Therefore, the present study was designed to assess the specific effects of excess corticosterone on Leydig cell NADPH generating enzymes and glucose oxidation. Adult Wistar rats (200-250 g, b.w.) were treated with corticosterone-21-acetate (2 mg/ 100 g, b.w., i.m., twice daily) for 10 days and corticosterone-21-acetate plus luteinizing hormone (LH) (100 microg/kg b.w., i.m., daily) for 10 days. After the treatment period, experimental animals and controls were killed, blood was collected and the sera separated for testosterone assay. Testes were removed and Leydig cells were isolated, purified and used for estimating the specific activity of NADPH generating enzymes and 14C-glucose oxidation. Serum testosterone, Leydig cellular 14C-glucose oxidation and the specific activities of 6-phosphogluconate dehydrogenase (6-PGDH), NADP-isocitrate dehydrogenase (ICDH) and malic enzyme were significantly decreased in corticosterone-treated rats. Co-administration of LH with corticosterone maintained the specific activities of 6-PGDH and ICDH and 14C-glucose oxidation at control level. Nevertheless, serum testosterone and Leydig cellular malic enzyme activity showed a significant decrease in these rats. In conclusion, the inhibitory effects of excess corticosterone on Leydig cell steroidogenesis are mediated through impaired glucose oxidation and defective NADPH generation. Co-administration of LH with corticosterone failed to prevent the decrease in serum testosterone and Leydig cell malic enzyme activity, suggesting the dominant inhibitory effects of excess corticosterone.

Culture of In Vitro Mouse Embryos with Vitamin E Improves Development.

To investigate the effects of vitamin E on the in vitro development of mouse embryos from 1-cell to blastocysts, embryos were cultured in two media, Brinster's BMOCIII and BMOCIII supplemented with 100 μM vitamin E (BMOCIIIvitE). The incorporation and oxidation of glucose were also compared between the embryos cultured in these media. Both the number of embryos that developed to the 1-cell stage and to the blastocyst stage were much larger for BMOCIIIvitE than BMOCIII. No significant differences in the incorporation of 14C-glucose or in the oxidation of 14C-glucose were found between the 2-cell embryos incubated in BMOCIIIvitE and BMOCIII. On the other hand, the incorporation and oxidation rates of 14C-glucose were significantly higher (p<0.05) in the embryos cultured in BMOCIIIvitE compared with those in BMOCIII at the blastocyst stage. These findings indicate that vitamin E has a beneficial effect on embryo development in mice, perhaps through protecting the cells from oxygen radicals in vitro.