High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32

We investigated the polyol pathway activity and the gene expression profiles in immortalized adult mouse Schwann cells (IMS32) under normal (5.6 mM) and high (30 and 56 mM) glucose conditions for 7-14 days in culture. Messenger RNA and the protein expression of aldose reductase (AR) and the intracellular sorbitol and fructose contents were up-regulated in IMS32 under high glucose conditions compared with normal glucose conditions. By employing DNA microarray and subsequent RT-PCR/northern blot analyses, we observed significant up-regulation of the mRNA expressions for serum amyloid A3 (SAA3), angiopoietin-like 4 (ANGPTL4) and ecotropic viral integration site 3 (Evi3), and the down-regulation of aldehyde reductase (AKR1A4) mRNA expression in the cells under high glucose (30 mM) conditions. The application of an AR inhibitor, SNK-860, to the high glucose medium ameliorated the increased sorbitol and fructose contents and the reduced AKR1A4 mRNA expression, while it had no effect on mRNA expressions for SAA3, ANGPTL4 or Evi3. Considering that the exposure to the high glucose (>or= 30 mM) conditions mimicking hyperglycaemia in vivo accelerated the polyol pathway in IMS32, but not in other previously reported Schwann cells, the culture system of IMS32 under those conditions may provide novel findings about the polyol pathway-related abnormalities in diabetic neuropathy.     

Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

Patients with diabetes have an increased risk of vascular complications. Suv39h1, a histone methyltransferase, plays a protective role against myocardial injury in diabetes. Herein, we intend to explore whether Suv39h1 could affect neointimal formation after vascular injury in diabetic rats and reveal the underlying mechanism. In this study, we generated adenovirus expressing Suv39h1 as well as lentivirus expressing Suv39h1‐targeting shRNA and evaluated the significance of Suv39h1 in vascular smooth muscle cells (VSMCs) under diabetic conditions. In vitro, we examined proliferative and migratory behaviours as well as the underlying signalling mechanisms in VSMCs in response to high glucose treatment. In vivo, we induced diabetes in SD rats with streptozocin and established the common carotid artery balloon injury model. Suv39h1 was found to be both necessary and sufficient to promote VSMC proliferation and migration under high glucose conditions. We observed corresponding changes in intracellular signalling molecules including complement C3 and phosphor‐ERK1/2. However, either up‐regulating or down‐regulating Suv39h1, phosphor‐p38 level was not significantly affected. Consistently, Suv39h1 overexpression led to accelerated neointima formation, while knocking down Suv39h1 reduced it following carotid artery injury in diabetic rats. Using microarray analyses, we showed that altering the Suv39h1 level in vivo dramatically altered the expression of myriad genes mediating different biological processes and molecular function. This study reveals the novel role of Suv39h1 in VSMCs of diabetes and suggests its potential role as a therapeutic target in diabetic vascular injury.     

Oxidative stress and cannabinoid receptor expression in type‐2 diabetic rat pancreas following treatment with Δ9‐THC

The objectives of study were (a) to determine alteration of feeding, glucose level and oxidative stress and (b) to investigate expression and localization of cannabinoid receptors in type‐2 diabetic rat pancreas treated with Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC). Rats were randomly divided into four groups: control, Δ⁹‐THC, diabetes and diabetes + Δ⁹‐THC groups. Diabetic rats were treated with a single dose of nicotinamide (85 mg/kg) 15 min before injection of streptozotocin (65 mg/kg). Δ⁹‐THC was administered intraperitoneally at 3 mg/kg/day for 7 days. Body weights and blood glucose level of rats in all groups were measured on days 0, 7, 14 and 21. On day 15 after the Δ⁹‐THC injections, pancreatic tissues were removed. Blood glucose levels and body weights of diabetic rats treated with Δ⁹‐THC did not show statistically significant changes when compared with the diabetic animals on days 7, 14 and 21. Treatment with Δ⁹‐THC significantly increased pancreas glutathione levels, enzyme activities of superoxide dismutase and catalase in diabetes compared with non‐treatment diabetes group. The cannabinoid 1 receptor was found in islets, whereas the cannabinoid 2 receptor was found in pancreatic ducts. Their localization in cells was both nuclear and cytoplasmic. We can suggest that Δ⁹‐THC may be an important agent for the treatment of oxidative damages induced by diabetes. However, it must be supported with anti‐hyperglycaemic agents. Furthermore, the present study for the first time emphasizes that Δ⁹‐THC may improve pancreatic cells via cannabinoid receptors in diabetes. The aim of present study was to elucidate the effects of Δ⁹‐THC, a natural cannabinoid receptor agonist, on the expression and localization of cannabinoid receptors, and oxidative stress statue in type‐2 diabetic rat pancreas. Results demonstrate that the cannabinoid receptors are presented in both Langerhans islets and duct regions. The curative effects of Δ⁹‐THC can be occurred via activation of cannabinoid receptors in diabetic rat pancreas. Moreover, it may provide a protective effect against oxidative damage induced by diabetes. Thus, it is suggested that Δ⁹‐THC can be a candidate for therapeutic alternatives of diabetes symptoms. Copyright © 2014 John Wiley & Sons, Ltd.     

Control of glyceroneogenic activity in rat brown adipose tissue

Brown adipose tissue (BAT) glyceroneogenesis was evaluated in rats either fasted for 48 h or with streptozotocin-diabetes induced 3 days previously or adapted for 20 days to a high-protein, carbohydrate-free (HP) diet, conditions in which BAT glucose utilization is reduced. The three treatments induced an increase in BAT glyceroneogenic activity, evidenced by increased rates of incorporation of [1-14C]pyruvate into triacylglycerol (TAG)-glycerol in vitro and a marked, threefold increase in the activity of BAT phosphoenolpyruvate carboxykinase (PEPCK). BAT glycerokinase activity was not significantly affected by fasting or diabetes. After unilateral BAT denervation of rats fed either the HP or a balanced diet, glyceroneogenesis activity increased in denervated pads, evidenced by increased rates of nonglucose carbon incorporation into TAG-glycerol in vivo (difference between 3H2O and [14C]glucose incorporations) and of [1-14C]pyruvate in vitro. PEPCK activity was not significantly affected by denervation. The data suggest that BAT glyceroneogenesis is not under sympathetic control but is sensitive to hormonal/metabolic factors. In situations of reduced glucose use there is an increase in BAT glyceroneogenesis that may compensate the decreased generation of glycerol-3-phosphate from the hexose.     

Fuzi Attenuates Diabetic Neuropathy in Rats and Protects Schwann Cells from Apoptosis Induced by High Glucose

Radix aconite lateralis preparata (Fuzi), a folk medicine, has long been used for the treatment of diabetes and paralysis in China. We examined the effect of Fuzi alone on diabetic rats and Schwann cells in high glucose and the components responsible for its activity. The major constituents of FZE were identified by HPLC-MS/MS data. Male Sprague Dawley rats (n = 36) were randomly divided into control, diabetic, FZE 1.75 g/kg, FZE 3.50 g/kg, FZE 7.00 g/kg, and methylcobalamin groups. After two weeks treatment, nerve conduction velocity and paw withdrawal latency were measured. In vitro, the Schwann cells were grouped according to exposure: normal glucose (NG), normal glucose plus mannitol (NG+M), high glucose (HG), and HG plus different concentrations of FZE (0.1 µg/ml, 1.0 µg/ml, and 10.0 µg/ml). Oxygen free radicals and apoptosis were evaluated through DCFH₂DA, DHE and annexin-PE/7-AAD assay, respectively. Apoptosis factors (Bax, Bcl-2, CytoC, caspase-3, and caspase-9) were analyzed using immunofluorescence. Nine alkaloids were identified. The results from animal model showed that FZE was effective in accelerating nerve conduction velocity and shortening paw withdrawal latency in diabetic rats. And in vitro, FZE was also found to protect Schwann cells against high glucose injury. FZE could significantly decrease the apoptotic ratio, superoxide anion and peroxide level. Furthermore, the apoptosis factors, including Bax, Bcl-2, CytoC, caspase-3, and caspase-9 were ameliorated in FZE treated groups. The HPLC-MSⁿ method is simple and suitable for the identification of alkaloids in Fuzi. FZE has a protective effect in diabetic neuropathic rats, which is probably achieved by the antiapoptotic effect of FZE on Schwann cells. Apoptosis factor data imply that FZE protected Schwann cells through the mitochondria pathway. Alkaloids are major components contributing to the protective effect.     

Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22^phox, NOX4, and p67^phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22^phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22^phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22^phox, NOX4, p67^phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.     

Late migration and seawater entry is physiologically disadvantageous for American shad juveniles

Juvenile American shad Alosa sapidissima were subjected to isothermal transfers into sea water (salinity 24)‘early’(1 September; 24° C) and ‘late’(10 November; 10° C) in the autumn migratory season. Early acclimation resulted in a modest osmotic perturbation that recovered rapidly. Haematocrit declined by 14% at 24 h, recovering within 48 h. Plasma osmolality increased by 6% at 4 h, recovering within 8 h. Early acclimation caused a two‐fold increase in gill Na⁺, K⁺‐ATPase activity by 24 h and a four‐fold increase by 4 days. The number of chloride cells on the primary gill filament increased two‐fold by 4 days. Chloride cells on the secondary lamellae rapidly decreased from 22 cells mm^?1 to <2 cells mm^?1 within 4 days. Late acclimation resulted in a severe and protracted osmotic perturbation. Haematocrit levels declined by 23% at 4 days, recovering by 14 days. Plasma osmolality increased by 36% by 48 h, recovering by 4 days. Initial gill Na⁺, K⁺‐ATPase activity was two‐fold greater than in ‘early’ fish and did not change during acclimation. Initial numbers of chloride cells on the primary filament were two‐fold greater than ‘early’ fish and did not increase during acclimation. Initial number of chloride cells on the secondary lamellae was five‐fold greater than ‘early’ fish (116 v. 22 cells mm^?1) and declined to negligible numbers over 14 days. Differences between initial measures for ‘early’ and ‘late’ fish reflect previously described physiological changes associated with migration. These data indicate that late migrants face a greater physiological challenge during seawater acclimation than early migrants. Physiological performance apparently limits the observed duration of autumnal migration.     

ENVIRONMENTAL EFFECTS ON EXOPOLYMER PRODUCTION BY MARINE BENTHIC DIATOMS: DYNAMICS,CHANGES IN COMPOSITION,AND PATHWAYS OF PRODUCTION1

Marine benthic diatoms excrete large quantities of extracellular polymeric substances (EPS), both as a function of their motility system and as a response to environmental conditions. Diatom EPS consists predominantly of carbohydrate‐rich polymers and is important in the ecology of cells living on marine sediments. Production rates, production pathways, and monosaccharide composition of water‐soluble (colloidal) carbohydrates, EPS, and intracellular storage carbohydrate (glucans) were investigated in the epipelic (mud‐inhabiting) diatoms Cylindrotheca closterium (Ehrenburg), Navicula perminta (Grün.) in Van Heurck, and Amphora exigua Greg. under a range of experimental conditions simulating aspects of the natural environment. Cellular rates of colloidal carbohydrate, EPS, and glucan production were significantly higher during nutrient‐replete compared with nutrient‐limited growth for all three species. The proportion of EPS in the extracellular carbohydrate pool increased significantly (to 44%–69%) as cells became nutrient limited. Cylindrotheca closterium produced two types of EPS differing in sugar composition and production patterns. Nutrient‐replete cells produced a complex EPS containing rhamnose, fucose, xylose, mannose, galactose, glucose, and uronic acids. Nutrient‐limited cells produced an additional EPS containing mannose, galactose, glucose, and uronic acids. Both EPS types were produced under illuminated and darkened conditions. ¹⁴C‐labeling revealed immediate production of ¹⁴C‐glucan and significant increases in ¹⁴C‐EPS between 3 and 4 h after addition of label. The glucan synthesis inhibitor 2,6‐dichlorobenzonitrile significantly reduced ¹⁴C‐colloidal carbohydrate and ¹⁴C‐EPS. The glucanase inhibitor P‐nitrophenyl β‐d ‐glucopyranoside resulted in accumulation of glucan within cells and lowered rates of ¹⁴C‐colloidal and ¹⁴C‐EPS production. Cycloheximide prevented glucan catabolism, but glucan production and EPS synthesis were unaffected.     

Insulin inhibits the JNK mediated cell death via upregulation of AKT expression in Schwann cells grown in hyperglycemia

Background

Schwann cells (SCs) are the glial cells of the peripheral nervous system, which forms a thick insulating structure around the axons. Hyperglycemia is known physiologic conditions in both type I and type II diabetes which causes diabetic neuropathy. But the SC possesses insulin receptors even though glucose uptake is independent of insulin. Since the insulin level is highly altered in diabetes, it is of greater importance to evaluate their role in the Schwann cell survival and death.

Methods

Schwann cells were isolated from neonatal pups and grown with and without insulin in hyperglycemic medium to mimic diabetic condition for 24 and 48 h. We studied the cell viability using 3 (4,5-dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide (MTT) and mitochondrial membrane potential (MMP) assay at different time interval on SCs. We also studied the protein and gene expression of Protein Kinase B (AKT) and Jun N-terminal kinase (JNK), which are greatly involved in cell survival and cell death respectively.

Results

The result shows that, high glucose levels for 48 h decrease the SC viability. Hyperglycemic condition induces the SC death by increasing the JNK expression which in turn reduces the MMP of glial cells. However, insulin administration for SCs grown in high glucose condition can reduce the JNK expression by activating AKT signaling pathway.

Conclusion

These observations demonstrate that the proper insulin balance is required for Schwann cells survival in hyperglycemic condition. Therefore, altered insulin signaling can be one of the reasons for demyelination of peripheral neurons in diabetic neuropathy.

     

Endothelial cells' biophysical,biochemical, and chromosomal aberrancies in high‐glucose condition within the diabetic range

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia‐induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells—especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high‐glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high‐glucose condition, the dynamic of the fatty acid profile—including polyunsaturated, monounsaturated, and saturated fatty acids—was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31⁺/CD34⁺) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell‐cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)‐1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold‐coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.     

Thyroid hormone T3 counteracts STZ induced diabetes in mouse

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrificed. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.     

Poly(ADP-ribose) polymerase 1 is involved in glucose toxicity through SIRT1 modulation in HepG2 hepatocytes

Accelerated glucose metabolism leads to oxidative stress and DNA damage in cells; these effects are related to glucose toxicity. The precise mechanisms of glucose toxicity are still unclear. The aim of this work was to investigate the mechanism of poly(ADP‐ribose) polymerase 1 (PARP1), which is a DNA repair enzyme activated by high‐glucose‐induced oxidative stress, and its effect on glucose toxicity in HepG2 hepatocytes. HepG2 cells were cultured under normal (5.5 mM) or high (30 mM) glucose conditions for 4 days. PJ34, which is an inhibitor of PARP1, was used to determine the downstream effects of PARP1 activation. PARP1 activity in 30 mM‐glucose‐treated cells was more than that in 5.5 mM‐glucose‐treated cells, and the activity correlated with the increase in ROS generation and DNA damage. PJ34 suppressed PARP1 activation and prevented the high‐glucose‐induced suppression of SIRT1 and AMP‐activated protein kinase (AMPK) activity, which was similar to its effect on the restoration of intracellular nicotinamide adenine dinucleotide (NAD) content. Further, the phosphorylation of insulin receptor was attenuated in response to insulin stimulation under high glucose conditions, and PJ34 could reverse this effect. The results of transfection of HepG2 cells with PARP1 small interfering RNA were similar to those obtained by treatment of the cells with PARP1 inhibitor PJ34. These data suggest that high‐glucose‐induced PARP1 activation might play a role in glucose toxicity by down‐regulating SIRT1 and AMPK activity through NAD depletion and resulting in insulin insensitivity. J. Cell. Biochem. 112: 299–306, 2011. © 2010 Wiley‐Liss, Inc.     

Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury

Hypoxia/reoxygenation (H/R)‐induced injury is the key factor associated with islet graft dysfunction. This study aims to examine the effect of mesenchymal stem cells (MSCs) on islet survival and insulin secretion under H/R conditions. Islets from rats were isolated, purified, cultured with or without MSCs, and exposed to hypoxia (O₂ ≤ 1%) for 8 h and reoxygenation for 24 and 48 h, respectively. Islet function was evaluated by measuring basal and glucose‐stimulated insulin secretion (GSIS). Apoptotic islet cells were quantified using Annexin V‐FITC. Anti‐apoptotic effects were confirmed by mRNA expression analysis of hypoxia‐resistant molecules, HIF‐1α, HO‐1, and COX‐2, using semi‐quantitative retrieval polymerase chain reaction (RT‐PCR). Insulin expression in the implanted islets was detected by immunohistological analysis. The main results show that the stimulation index (SI) of GSIS was maintained at higher levels in islets co‐cultured with MSCs. The MSCs protected the islets from H/R‐induced injury by decreasing the apoptotic cell ratio and increasing HIF‐1α, HO‐1, and COX‐2 mRNA expression. Seven days after islet transplantation, insulin expression in the MSC‐islets group significantly differed from that of the islets‐alone group. We proposed that MSCs could promote anti‐apoptotic gene expression by enhancing their resistance to H/R‐induced apoptosis and dysfunction. This study provides an experimental basis for therapeutic strategies based on enhancing islet function. Copyright © 2010 John Wiley & Sons, Ltd.     

Effect of cerebrocrast, a new long-acting compound on blood glucose and insulin levels in rats when administered before and after STZ-induced diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that is characterized by selective destruction of insulin secreting pancreatic islets beta-cells. The formation of cytokines (IL-1beta, IL-6, TNF-alpha, etc.) leads to extensive morphological damage of beta-cells, DNA fragmentation, decrease of glucose oxidation, impaired glucose-insulin secretion and decreased insulin action and proinsulin biosynthesis. We examined the protective effect of a 1,4-dihydropyridine (DHP) derivative cerebrocrast (synthesized in the Latvian Institute of Organic Synthesis) on pancreatic beta-cells in rats possessing diabetes induced with the autoimmunogenic compound streptozotocin (STZ). Cerebrocrast administration at doses of 0.05 and 0.5 mg/kg body weight (p.o.) 1 h or 3 days prior to STZ as well as at 24 and 48 h after STZ administration partially prevented pancreatic beta-cells from the toxic effects of STZ, and delayed the development of hyperglycaemia. Administration of cerebrocrast starting 48 h after STZ-induced diabetes in rats for 3 consecutive days at doses of 0.05 and 0.5 mg/kg body weight (p.o.) significantly decreased blood glucose level, and the effect remained 10 days after the last administration. Moreover, in these rats, cerebrocrast evoked an increase of serum immunoreactive insulin (IRI) level during 7 diabetic days as compared to both the control normal rats and the STZ-induced diabetic control rats. The STZ-induced diabetic rats that received cerebrocrast had a significantly high serum IRI level from the 14th to 21st diabetic days in comparison with the STZ-induced diabetic control.The IRI level in serum as well as the glucose disposal rate were significantly increased after stimulation of pancreatic beta-cells with glucose in normal rats that received cerebrocrast, administered 60 min before glucose. Glucose disposal rate in STZ-induced diabetic rats as a result of cerebrocrast administration was also increased in comparison with STZ-diabetic control rats. Administration of cerebrocrast in combination with insulin intensified the effect of insulin. The hypoglycaemic effect of cerebrocrast primarily can be explained by its immunomodulative properties. Moreover, cerebrocrast can act through extrapancreatic mechanisms that favour the expression of glucose transporters, de novo insulin receptors formation in several cell membranes as well as glucose uptake.     

Environmental salinity-modified osmoregulatory response in the sub-Antarctic notothenioid fish Eleginops maclovinus

In this study we assessed changes in the osmoregulatory system of juvenile sub-Antarctic Eleginops maclovinus submitted to different environmental salinities (5, 15, 32 and 45 psu) using two different acclimation trials: (1) an end-point experiment (exposure for 14 days) and (2) a time course experiment (specimens were sampled on days 1, 3, 7 and 14 post-transfer). Plasma osmolality, cortisol and metabolites (glucose, lactate and protein) values as well as Na⁺, K⁺-ATPase (NKA) activity were assessed in several osmoregulatory tissues (gills, kidney and intestine). In both trials, acclimation to different environmental salinities for 14 days induced changes in plasma metabolites (glucose, lactate and proteins) as well as cortisol values related to salinity challenges. Plasma osmolality and gill NKA activity presented a direct and positive relationship with respect to environmental salinity, while kidney NKA activity showed a “U-shaped” relationship. Anterior intestinal NKA activity increased in response to environmental salinity and apparently did not change in the middle portion of this organ, while it was enhanced in the posterior portion in environmental salinities different than seawater. Plasma metabolite values increased under hypo- and hypersaline conditions, indicating the importance of these energy substrates in extreme environments. The time course study revealed that specimens of E. maclovinus are able to accommodate their osmotic and metabolic system to respond to osmoregulatory challenges by allostatic changes.     

Change of Beclin‐1 dependent on ATP, [Ca2+]i and MMP in PC12 cells following oxygen‐glucose deprivation‐reoxygenation injury

Autophagy is usually up‐regulated to provide more ATP in response to starvation or OGD (oxygen‐glucose deprivation), but the relationship between autophagy and ATP, [Ca²⁺]_i (intracellular free Ca²⁺ concentration) or MMP (mitochondrial membrane potential) during reoxygenation is not yet fully clear. The role of autophagy is unknown in PC12 cells subjected to 2 h OGD with different time points of reoxygenation. In the present study, we showed that Beclin‐1 was up‐regulated beginning at 0 h reoxygenation peaking at 24 h and lasting for 48 h. Cell viability was decreased from 0 to 48 h reoxygenation, reaching its minimum at 10 h reoxygenation. ATP was decreased from 0 to 10 h reoxygenation, reaching its minimum at 4 h reoxygenation. A significant negative correlation was observed between ATP and Beclin‐1 (r = ?0.61, P<0.05) at 0 h reoxygenation, but ATP was not significant related (r = 0.24, P>0.05) to Beclin‐1 at 24 h reoxygenation. Besides, Nimodipine, a calcium antagonist, significantly reduced [Ca²⁺]_i and Beclin‐1, but increased MMP in OGD/R‐treated cells. At 24 h reoxygenation, Beclin‐1 expression reached its maximum, cell viability continued to increase, and ATP was higher than that before OGD. These results suggest that energy metabolism dysfunction can induce autophagy during OGD in PC12 cells. Increased [Ca²⁺]_i and decreased MMP may induce autophagy during reoxygenation in PC12 cells. Autophagy may be a protective effect on PC12 cells treated with different time points of reoxygenation after 2 h OGD.     

Cyclooxygenase‐2 over‐expression inhibits liver apoptosis induced by hyperglycemia

Increased expression of COX‐2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX‐2 protects hepatocytes from several pro‐apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX‐2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX‐2 (hCOX‐2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post‐injection, Wt diabetic animals showed a decrease in PI3K activity and P‐Akt levels, an increase of P‐JNK, P‐p38, pro‐apoptotic Bad and Bax, release of cytochrome c and activities of caspases‐3 and ‐9, leading to an increased apoptotic index. This situation was improved in diabetic COX‐2 Tg. In addition, SID COX‐2 Tg showed increased expression of anti‐apoptotic Mcl‐1 and XIAP. Pro‐apoptotic state in the liver of diabetic animals was improved by over‐expression of COX‐2. We also analyzed the roles of high glucose‐induced apoptosis and hCOX‐2 in vitro. Non‐transfected and hCOX‐2‐transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non‐transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX‐2‐transfected cells was suppressed by addition of DFU (COX‐2 selective inhibitor), and mimicked by addition of PGE₂ in non‐transfected cells. Taken together, these results demonstrate that hyperglycemia‐induced hepatic apoptosis is protected by hCOX‐2 expression. J. Cell. Biochem. 114: 669–680, 2013. © 2012 Wiley Periodicals, Inc.     

Preparation of radioactive diacetyl,acetoin, and 2,3-butylene glycol

Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[3-¹⁴C]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 × 10⁴ cpm/μmol and acetoin with specific activity of 6.8 × 10⁴ cpm/μmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [¹⁴C]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 × 10⁴ cpm/μmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.     

Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells induced by high glucose concentration

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.