Glucose-cultured Cells Research Articles

Long term high glucose exposure induces premature senescence in retinal endothelial cells.

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated β-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated β-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated β-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

Acetate Promotes a Differential Energy Metabolic Response in Human HCT 116 and COLO 205 Colon Cancer Cells Impacting Cancer Cell Growth and Invasiveness.

Under dysbiosis, a gut metabolic disorder, short-chain carboxylic acids (SCCAs) are secreted to the lumen, affecting colorectal cancer (CRC) development. Butyrate and propionate act as CRC growth inhibitors, but they might also serve as carbon source. In turn, the roles of acetate as metabolic fuel and protein acetylation promoter have not been clearly elucidated. To assess whether acetate favors CRC growth through active mitochondrial catabolism, a systematic study evaluating acetate thiokinase (AcK), energy metabolism, cell proliferation, and invasiveness was performed in two CRC cell lines incubated with physiological SCCAs concentrations. In COLO 205, acetate (+glucose) increased the cell density (50%), mitochondrial protein content (3–10 times), 2-OGDH acetylation, and oxidative phosphorylation (OxPhos) flux (36%), whereas glycolysis remained unchanged vs. glucose-cultured cells; the acetate-induced OxPhos activation correlated with a high AcK activity, content, and acetylation (1.5–6-fold). In contrast, acetate showed no effect on HCT116 cell growth, OxPhos, AcK activity, protein content, and acetylation. However, a substantial increment in the HIF-1α content, HIF-1α-glycolytic protein targets (1–2.3 times), and glycolytic flux (64%) was observed. Butyrate and propionate decreased the growth of both CRC cells by impairing OxPhos flux through mitophagy and mitochondrial fragmentation activation. It is described, for the first time, the role of acetate as metabolic fuel for ATP supply in CRC COLO 205 cells to sustain proliferation, aside from its well-known role as protein epigenetic regulator. The level of AcK determined in COLO 205 cells was similar to that found in human CRC biopsies, showing its potential role as metabolic marker.

Hyperglycemia modulates M1/M2 macrophage polarization via reactive oxygen species overproduction in ligature-induced periodontitis.

Periodontitis in diabetic patients is characterized by enhanced inflammation and aggravated tissue damage in comparison with that in non-diabetic counterparts. The progression of periodontal damage under diabetic condition can be partly ascribed to hyperglycemia-induced disturbance between immune activation and inflammation resolution, where macrophages are capable of participating given their plasticity in response to different stimuli. Herein, we aimed to investigate the changes of macrophage polarization in periodontitis under diabetic condition and the underlying mechanism. Type-1 diabetes was induced by the injection of streptozotocin (STZ, 60mg/kg) in Sprague-Dawley rats. Rats in N-acetyl cysteine (NAC)-treated groups received NAC dissolved in drinking water (200mg/kg/day). Experimental periodontitis was induced by ligating 3-0 silk around left maxillary second molars for 4weeks. Alveolar bone destruction was tested by micro-computed tomography and tartrate-resistant acid phosphatase (TRAP) staining. M1/M2 macrophage polarization in periodontal tissue was detected by immunohistochemistry staining. RAW264.7 were cultured in normal glucose (5.5mM) or high glucose environment (25mM) with or without NAC (8mmol/L). LPS (100ng/ml) and IL-4 (20ng/ml) were used to induce M1macrophages and M2 macrophages, respectively. M1/M2 macrophage polarization was detected by qRT-PCR, immunofluorescent staining, and flow cytometry. Reactive oxygen species (ROS) accumulation was detected by fluorogenic probes. RANKL (100ng/ml) were applied to induce osteoclastogenic differentiation of RAW264.7, and osteoclast formation was examined by TRAP staining. Rats with diabetes displayed enhanced macrophages infiltration and M1 macrophage polarization in periodontal lesions compared with vehicle-treated rats. Under LPS or IL-4 stimulation, high glucose culture of RAW264.7 elevated ROS level and increased the expression of M1 macrophage markers (iNOS, TNF-α, and IL-6) whereas decreased the expression of M2 macrophage markers (Arg-1 and CD206). Supernatants of high glucose-treated M1/M2 macrophages enhanced osteoclast formation compared to normal glucose-cultured cells. Decreasing ROS level via NAC partially reversed the effect of high glucose on M1/M2 macrophage polarization. Meanwhile, daily intake of NAC in rodent models inhibited M1 macrophage polarization, which subsequently ameliorated alveolar bone loss and decreased osteoclast numbers in periodontitis in diabetic rats. These findings demonstrated that hyperglycemia could polarize macrophage toward M1 macrophages via overproducing ROS under inflammatory condition, which might take responsibility for aggravated periodontal damage in periodontitis under diabetic condition. Inhibiting M1 macrophages and restoring M2 macrophages by ROS scavenger is hopefully a potential adjunct treatment strategy for diabetic periodontitis.

Interleukin 6 Function in the Skin and Isolated Keratinocytes Is Modulated by Hyperglycemia.

Diabetes currently affects over twenty-five million Americans. Annual health care cost of diabetes exceeds $254 billion and is associated with a distinct set of diabetic complications that include delayed wound healing and diabetic ulcers. Interleukin 6 (IL-6) plays an important role in wound healing and is known to be elevated in the serum of both type I and type II diabetes patients. This study assesses the expression and function of IL-6 in the hyperglycemic epidermis and keratinocyte culture. Streptozotocin-treated mice were wounded six weeks after induction of hyperglycemia. Wound closure, protein, and mRNA expression were assessed up to 13 days of postwounding. Wound closure was delayed 4-5 days in hyperglycemic animals. Hyperglycemic wounds displayed greater IL-6 and IL-6Rα protein expression at 1, 7, and 10 days of postwounding compared to euglycemic control. However, IL-6Rα mRNA expression was reduced at all time points beyond day 1, while IL-6 mRNA expression did not significantly differ at any time point. SOCS3 mRNA expression was higher in the hyperglycemic skin at every time point. Imaging of fluorescent immunohistology also revealed significantly lower expression of SOCS3, but higher nuclear pSTAT3 in the epidermis of the hyperglycemic skin. Primary mouse keratinocytes cultured in high glucose for 7 days displayed 2-fold higher IL-6Rα mRNA and higher rmIL-6-induced nuclear pSTAT3, but lower SOCS3 basal levels compared to normal glucose-cultured cells. Thus, it appears that delayed diabetic skin wound healing is associated with increased induction and expression of IL-6 and its receptor, but its function in epidermal keratinocytes may be impaired.

Short-term high glucose culture potentiates pancreatic beta cell function

The exposure of pancreatic islets to high glucose is believed to be one of the causal factors of the progressive lowering of insulin secretion in the development of type 2 diabetes. The progression of beta cell failure to type 2 diabetes is preceded by an early positive increase in the insulin secretory response to glucose, which is only later followed by a loss in the secretion capacity of pancreatic islets. Here we have investigated the electrophysiological mechanisms underlying the early glucose-mediated gain of function. Rodent pancreatic islets or dispersed islet cells were cultured in medium containing either 5.6 (control) or 16.7 (high-glucose) mM glucose for 24 h after isolation. Glucose-stimulated insulin secretion was enhanced in a concentration-dependent manner in high glucose-cultured islets. This was associated with a positive effect on beta cell exocytotic capacity, a lower basal KATP conductance and a higher glucose sensitivity to fire action potentials. Despite no changes in voltage-gated Ca2+ currents were observed in voltage-clamp experiments, the [Ca2+]I responses to glucose were drastically increased in high glucose-cultured cells. Of note, voltage-dependent K+ currents were decreased and their activation was shifted to more depolarized potentials by high-glucose culture. This decrease in voltage-dependent K+ channel (Kv) current may be responsible for the elevated [Ca2+]I response to metabolism-dependent and independent stimuli, associated with more depolarized membrane potentials with lower amplitude oscillations in high glucose-cultured beta cells. Overall these results show that beta cells improve their response to acute challenges after short-term culture with high glucose by a mechanism that involves modulation not only of metabolism but also of ion fluxes and exocytosis, in which Kv activity appears as an important regulator.

Increased sorbitol levels in the hypertrophic ligamentum flavum of diabetic patients with lumbar spinal canal stenosis.

The pathomechanism of the ligamentum flavum (LF) hypertrophy in diabetic patients with lumbar spinal canal stenosis (LSCS) remains unclear. A cross-sectional study was undertaken to investigate the mechanism of LF hypertrophy in these patients. Twenty-four diabetic and 20 normoglycemic patients with LSCS were enrolled in the study. The structure of the LF in the study subjects was evaluated using histological and immunohistochemical methods, and the levels of sorbitol, pro-inflammatory cytokines, and the fibrogenic factor, TGF-β1, in the LF were analyzed. In vitro experiments were performed using NIH3T3 fibroblasts to evaluate the effect of high-glucose conditions and an aldose reductase inhibitor on the cellular production of sorbitol, pro-inflammatory factors, and TGF-β1. We found that the LF of diabetic patients exhibited significantly higher levels of sorbitol and pro-inflammatory cytokines, TGF-β1 and of CD68-positive staining than that of the normoglycemic subjects. The diabetic LF was significantly thicker than that of the controls, and showed evidence of degeneration. The high glucose-cultured fibroblasts exhibited significantly higher levels of sorbitol, pro-inflammatory factors, and TGF-β1 compared to the low glucose-cultured cells, and these levels were dose-dependently reduced by treatment with the aldose reductase inhibitor. Taken together, our data suggests that increased sorbitol levels in the LF of diabetic patients results in increased production of pro-inflammatory and fibrogenic factor, which contribute to LF hypertrophy, and could increase the susceptibility of diabetic patients to LSCS. Furthermore, aldose reductase inhibition effectively reduced the levels of sorbitol and sorbitol-induced pro-inflammatory factor expression in high glucose-cultured fibroblasts. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1058-1066, 2017.

Increased activity of mitochondrial uncoupling protein 2 improves stress resistance in cultured endothelial cells exposed in vitro to high glucose levels.

The endothelium is relatively independent of the mitochondrial energy supply, but mitochondria-derived ROS may play an important role in the development of many cardiovascular diseases. Energy-dissipating uncoupling proteins (UCPs) mediate free fatty acid-activated, purine nucleotide-inhibited proton conductance (uncoupling) in the inner mitochondrial membrane. We have described a functional characteristic and an antioxidative role for UCP2 in endothelial cells and isolated mitochondria and how this function is altered by long-term growth in high concentrations of glucose. Human umbilical vein endothelial cells (EA.hy926 line) were grown in media with either high (25 mM) or normal (5.5 mM) glucose concentrations. Under nonphosphorylating and phosphorylating conditions, UCP activity was significantly higher in mitochondria isolated from high glucose-treated cells. More pronounced control of the respiratory rate, membrane potential, and ROS by UCP2 was observed in these mitochondria. A greater UCP2-mediated decrease in ROS generation indicates an improved antioxidative role for UCP2 under high glucose conditions. Mitochondrial and nonmitochondrial ROS generations were significantly higher in high glucose-treated cells independent of UCP2 expression. UCP2 gene silencing led to elevated mitochondrial ROS formation and ICAM1 expression, especially in high glucose-cultured cells. UCP2 influenced endothelial cell viability and resistance to oxidative stress. Endothelial cells exposed to high glucose concentrations were significantly more resistant to peroxide. In these cells, the increased activity of UCP2 led to improved stress resistance and protection against acute oxidative stress. Our results indicate that endothelial UCP2 may function as a sensor and negative regulator of mitochondrial ROS production in response to hyperglycemia.

Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR) was significantly increased whereas extracellular acidification rate (ECAR), a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2– level compared to cells in the glucose model. An antimycin A (AA) dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a galactose media model and are consequently more susceptible to mitochondrial toxicants.

Dynamic localization and functional implications of C-peptide might for suppression of iNOS in high glucose-stimulated rat mesangial cells

Although C-peptide has unique beneficial effects on diabetic nephropathy (DN), its functional localization and molecular mechanism have not been fully clarified. Whether C-peptide exhibits its protective role through the regulation of inducible nitric oxide synthase (iNOS), a key enzyme in oxidative stress, is not clear. In this study, it was revealed that C-peptide could enter the nucleus of high glucose-stimulated mesangial cells, especially in a time-dependent manner by high glucose pretreatment, while no C-peptide was detected in low glucose-cultured mesangial cells. The dynamic functional localization of C-peptide might be the intrinsic cause of its unique beneficial effects for DN, which may provide a foundation for further clarification of its underlying mechanism. Our preliminarily data also shown C-peptide suppressed the iNOS expression. Taking together, these results revealed the dynamic functional localization of C-peptide by high glucose stimulation in rat mesangial cells, which might suppress expression of iNOS to exhibit its protective effects.

Estrogen Modulates Metabolic Pathway Adaptation to Available Glucose in Breast Cancer Cells

Most cancers use glucose as substrate for aerobic glycolysis in preference to oxidative phosphorylation. However, variable glucose concentrations within the in-vivo tumor microenvironment may necessitate metabolic plasticity. Furthermore, little information exists on a role for estrogen receptors in modulating possible metabolic adaptations in breast cancer cells. Here we find that MCF-7 cells switch between metabolic pathways depending on glucose availability and 17β-estradiol (E(2)) potentiates adaptation. In high glucose conditions E(2) up-regulates glycolysis via enhanced AKT kinase activity and suppresses tricarboxylic acid cycle activity. After a decrease in extracellular glucose, mitochondrial pathways are activated in preference to glycolysis. In this setting, E(2) suppresses glycolysis and rescues cell viability by stimulating the tricarboxylic acid cycle via the up-regulation of pyruvate dehydrogenase (PDH) activity. E(2) also increases ATP in low glucose-cultured cells, and the novel phosphorylation of PDH by AMP kinase is required for these metabolic compensations. Capitalizing on metabolic vulnerability, knockdown of PDH in the low-glucose state strongly potentiates ionizing radiation-induced apoptosis and reverses the cell survival effects of E(2). We propose that lowering glucose substrate and inhibiting PDH may augment adjuvant therapies for estrogen receptor-positive breast cancer.

High glucose increases nitric oxide generation in lipopolysaccharide-activated macrophages by enhancing activity of protein kinase C-α/δ and NF-κB

Although several mechanisms by which hyperglycemia modulate inflammation have been proposed, it remains unclear how hyperglycemia regulates inflammation induced by lipopolysaccharide (LPS). We hypothesized that hyperglycemia might interplay with LPS to modulate the generation of an inflammatory mediator. RAW 264.7 macrophages cultured in medium containing either normal glucose (5.5-mM) or high glucose (HG) (15- and 25-mM) were treated with LPS. The nitric oxide (NO) generation, inducible NO synthase (iNOS) expression and cytokine release were then quantified by Griess reaction, western blot, and enzyme-linked immunosorbent assay (ELISA) respectively. The effect of HG on the activation of kinase and Nuclear Factor-Kappa B (NF-κB) were measured by western blot and NF-κB reporter assay respectively. Without LPS stimulation, HG alone did not induce NO generation and cytokine secretion; but LPS-induced NO generation, iNOS expression, and interleukin-1beta (IL-1β) secretion were higher in HG-cultured cells than in normal glucose-cultured cells. In contrast, LPS-induced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) secretion were lower in HG-cultured cells than in normal glucose-cultured cells. Furthermore, HG increased iNOS expression and NO generation by enhancing phosphorylation levels of protein kinase C-alpha (PKC-α), protein kinase C-delta (PKC-δ), and p38 phosphorylation and NF-κB transcriptional activity. This study revealed a possible role of PKC-α and PKC-δ potentially involved in diabetes-promoted inflammation.

Nucleolar Follistatin Promotes Cancer Cell Survival under Glucose-deprived Conditions through Inhibiting Cellular rRNA Synthesis

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

Glucose Regulates Interleukin-8 Production in Aortic Endothelial Cells through Activation of the p38 Mitogen-activated Protein Kinase Pathway in Diabetes

We have shown that chronic elevated glucose (25 mm) increases monocyte adhesion to human aortic endothelial cells (EC). This increased adhesion is mediated primarily through induction of interleukin (IL)-8 via activation of the transcription factor AP-1 (Srinivasan, S., Yeh, M., Danziger, E. C., Hatley, M. E., Riggan, A. E., Leitinger, N., Berliner, J. A., and Hedrick, C. C. (2003) Circ. Res. 92, 371-377). In the current study, we identified the elements in the AP-1 transcriptional complex that are activated by glucose. These elements include c-Jun, c-Fos, and Fra-1. AP-1 is activated by cellular oxidative stress, and we have reported significant production of ROS by high glucose-cultured cells. We examined signaling pathways upstream of AP-1 in EC that lead to AP-1 activation by HG. EC cultured in 25 mm glucose had a 2-fold increase in p38 phosphorylation compared with control normal glucose-cultured EC. Inhibition of the p38 pathway using 5 microm SB203580 significantly reduced glucose-mediated IL-8 mRNA production by 60%. Furthermore, blocking p38 pathway activation using a dominant-negative p38 construct significantly reduced glucose-mediated monocyte adhesion by 50%. Thus, glucose-stimulated monocyte adhesion is primarily regulated through phosphorylation of p38 with subsequent activation of AP-1, leading to IL-8 production. To study this pathway in the setting of diabetes, we used the db/db mouse. P38 phosphorylation was increased in diabetic db/db mice compared with control mice. We found a dramatic elevation in plasma levels of KC, the mouse ortholog of IL-8 in diabetic db/db mice (1800 +/- 100 pg/ml KC in db/db versus 300 +/- 75 pg/ml in C57BL/6J control mice, p < 0.0001). Inhibition of the p38 pathway in diabetic db/db mice significantly reduced monocyte adhesion by 50%. Taken together, these data indicate that chronic elevated glucose in diabetes activates the p38 MAP kinase pathway to increase inflammatory IL-8 gene induction and monocyte/endothelial adhesion.

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.

Glucose starvation results in UDP-glucose deficiency and inactivation of glycogen synthase

The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to ∼10% of those in cells cultured with glucose. GS activity, protein and mRNA amounts in pyruvate-cultured cells were decreased to ∼35, 60, and 60%, respectively, of values in glucose-cultured cells. Incubation of extracts from glucose-cultured cells with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS. However, binding in immunoprecipitates from pyruvate-cultured cells was decreased to ∼25% of values in glucose-cultured cells. These data indicate that glucose starvation and the subsequent depletion of UDP-glucose result in: (1) inactivation of GS, owing to a decrease in its ability to bind UDP-glucose, and (2) decreased amount of GS protein, owing to a decrease in the levels of GS mRNA.

Aminoguanidine exerts a beta-cell function-preserving effect in high glucose-cultured beta-cells (INS-1).

We investigated the effects of aminoguanidine (AG) on β-cell functions in an insulin secreting cell line (INS-1). Culture with 27 mM glucose for one week markedly decreased both insulin release and insulin content compared to culture in 0.8 mM or 3.3 mM glucose. Relative to culture at 27 mM glucose alone, the co-exposure to 1 mM AG almost doubled basal as well as glucose or 25 mM KCl-stimulated insulin release and increased insulin content by 42%. AG failed to affect release and content in cells cultured at 0.8 or 3.3 mM glucose. Preproinsulin mRNA content in 27 mM glucose-cultured cells was 52% suppressed compared to 0.8 mM glucose-cultured cells, and AG treatment partially counteracted this decline. Advanced glycosylation end product (AGE)-associated fluorescence (370 nm excitation and 440 nm emission) of cells′ extracts did not differ between 27 mM and 0.8 mM glucose-cultured cells after 1 week of culture and fluorescence was unaffected by AG. Accumulation of nitrite into culture media was markedly increased from 27 mM glucose-cultured cells, and this accumulation was 33% suppressed by AG. In conclusion, AG partially protects against glucotoxic effects in INS-1 cells. These beneficial effects may involve a decrease in early glycation products and/or nitric oxide synthase (NOS) activity. The effects which were obtained after one week of high glucose exposure may supplement AGE-associated effects seen after chronically elevated glucose.

Sugar catabolism in Aquaspirillum gracile.

Aquaspirillum (Spirillum) gracile is one of the few spirilla that cause acidification of the medium when cultured with sugars. Acidic reactions have been reported only for d-glucose, d-galactose, and l-arabinose, and the mode of attack of these sugars has not been previously investigated. The soluble portion of extracts of glucose-cultured cells of A. gracile ATCC 19624 was found by spectrophotometric methods to contain enzyme activities characteristic of the Entner-Doudoroff and Embden-Meyerhof-Parnas pathways. No activity for 6-phosphogluconate dehydrogenase (EC 1.1.1.44) was detected. Pyridine nucleotide-linked dehydrogenase activities for l-arabinose and d-galactose (EC 1.1.1.46 and EC 1.1.1.48) occurred in the soluble fraction of cells cultured with either sugar. Glucose-cultured cells contained not only glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities but also glucose dehydrogenase (EC 1.1.1.47) activity. Enzymes capable of oxidizing gluconate were not detectable, but gluconokinase (EC 2.7.1.12) activity was present. Paper chromatographic analysis of the spent culture supernatant media from glucose-cultured cells indicated an accumulation of gluconic acid, and this was confirmed by enzymatic methods. Evidence is presented for the production of d-galactonic and l-arabonic acids in cultures containing d-galactose or l-arabinose, respectively.

Glucose transport in membrane vesicles from Azotobacter vinelandii, : Properties of the glucose carrier in situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate. The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K i of 9 μ m at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.