Hydrolysing the soluble protein secreted by Escherichia coli in trans-4-hydroxy-L-proline fermentation increased dissolve oxygen to promote high-level trans-4-hydroxy-L-proline production.

Trans-4-hydroxy-L-proline (Hyp) production by Escherichia coli (E. coli) in fermentation is a high-oxygen-demand process. E. coli secretes large amounts of soluble protein, especially in the anaphase of fermentation, which is an important factor leading to inadequate oxygen supply. And acetic acid that is the major by-product of Hyp production accumulates under low dissolved oxygen (DO). To increase DO and achieve high-level Hyp production, soluble protein was hydrolysed by adding protease in Hyp fermentation. The optimal protease, concentration, and addition time were trypsin, 0.2 g/L, and 18 h, respectively. With the addition of trypsin, the soluble protein in Hyp fermentation decreased by 43.5%. The DO could be maintained at 20-30% throughout fermentation. Hyp production and glucose conversion rate were 45.3 g/L and 18.1%, which were increases of 24.1% and 8.4%, respectively. The accumulation of acetic acid was decreased by 52.1%. The metabolic flux of Hyp was increased by 44.2% and the flux of acetate was decreased by 51.0%.

Acetylshikonin stimulates glucose uptake in L6 myotubes via a PLC-ß3/PKCδ-dependent pathway.

Acetylshikonin, a naphthoquinone derivative derived from Lithospermum erythrorhizon, has been shown to have various pharmacological activities; however, its effect on diabetes has rarely been reported. We investigated the hypoglycemic effect of acetylshikonin and found that it decreased blood glucose to a greater extent than insulin and improved glucose tolerance in mice. It also increased glucose uptake in L6 myotubes by inducing the expression and translocation of glucose transporter 4 via decomposition of phosphatidylinositol, increased generation of diacylglycerol, and activation of protein kinase C delta cascades; this is an insulin-, reactive oxygen species-, and AMP-activated protein kinase-independent pathway for glucose uptake. Our findings highlight the antidiabetic potential of acetylshikonin via a possible novel pathway for glucose uptake in L6 myotubes.

Anti-diabetic effect of three new norditerpenoid alkaloids in vitro and potential mechanism via PI3K/Akt signaling pathway.

Diabetes is a metabolic disease with the characteristic of high blood glucose (hyperglycemia). In our previous study, we found that nigelladines A-C (compounds A-C), three norditerpenoid alkaloids from the seeds of Nigella glandulifera Freyn (Ranunculaceae) exhibited protein of tyrosine phosphatase 1B (PTP1B) inhibitory activity in vitro. In the present study, we further investigated their anti-diabetes activities in L6 moytubes and illuminated the mechanisms of action of compounds A-C. Several parameters of glucose metabolism such as glucose consumption, glycogen content and hexokinase activity were increased by compounds A-C. The results suggested that compounds A-C improved glucose metabolism through promoting synthesis of glycogen. Expression of PTP1B protein was inhibited by compounds A-C in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by compounds A-C and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the insulin-mediated induction of insulin receptor substrate-1 (IRS-1) and glycogen synthase kinase-3ß (GSK-3ß) were also suppressed by wortmannin. Western blot results indicated that compounds A-C-induced IRS-1/Akt activation was likely a consequence of PTP1B inhibition. Compounds A-C promoted glycogen synthesis through Akt-mediated GSK3 phosphorylation. Therefore, activation of PI3K/Akt insulin signaling pathway and suppression of PTP1B is the molecular mechanism that contributes to the anti-diabetic effect of compounds A-C in cellular models. The three alkaloids potentially serve as lead compounds for the development of antidiabetic drugs.

Exposure to Concentrated Ambient PM2.5 Shortens Lifespan and Induces Inflammation-Associated Signaling and Oxidative Stress in Drosophila.

Exposure to ambient PM 2.5 is associated with human premature mortality. However, it has not yet been toxicologically replicated, likely due to the lack of suitable animal models. Drosophila is frequently used in longevity research due to many incomparable merits. The present study aims to validate Drosophila models for PM 2.5 toxicity study through characterizing their biological responses to exposure to concentrated ambient PM 2.5 (CAP). The survivorship curve demonstrated that exposure to CAP markedly reduced lifespan of Drosophila. This antilongevity effect of CAP exposure was observed in both male and female Drosophila, and by comparison, the male was more sensitive [50% survivals: 20 and 48 days, CAP- and filtered air (FA)-exposed males, respectively; 21 and 40 days, CAP- and FA-exposed females, respectively]. Similar to its putative pathogenesis in humans, CAP exposure-induced premature mortality in Drosophila was also coincided with activation of pro-inflammatory signaling pathways including Jak, Jnk, and Nf-κb and increased systemic oxidative stress. Furthermore, like in humans and mammals, exposure to CAP significantly increased whole-body and circulating glucose levels and increased mRNA expression of Ilp2 and Ilp5 , indicating that CAP exposure induces dysregulated insulin signaling in Drosophila. Similar to effects on humans exposure to CAP leads to premature mortality likely through induction of inflammation-associated signaling, oxidative stress, and metabolic abnormality in Drosophila, strongly supporting that it can be a useful model organism for PM 2.5 toxicity study.

Astragaloside IV facilitates glucose transport in C2C12 myotubes through the IRS1/AKT pathway and suppresses the palmitate-induced activation of the IKK/IκBα pathway.

Astragaloside IV is a monomer isolated from Astragalus membranaceus (Fisch.) Bunge, which is one of the most widely used plant-derived drugs in traditional Chinese medicine for diabetes therapy. In the present study, we aimed to examine the effects of astragaloside IV on glucose in C2C12 myotubes and the underlying molecular mechanisms responsible for these effects. Four-day differentiated C2C12 myotubes were exposed to palmitate for 16 h in order to establish a model of insulin resistance and 3H glucose uptake, using 2-Deoxy­D­[1,2-3H(N)]-glucose (radiolabeled 2-DG), was detected. Astragaloside IV was added 2 h prior to palmitate exposure. The translocation of glucose transporter 4 (GLUT4) was evaluated by subcellular fractionation, and the expression of insulin signaling molecules such as insulin receptor ß (IRß), insulin receptor substrate (IRS)1/protein kinase B (AKT) and inhibitory κB kinase (IKK)/inhibitor-κBα (IκBα), which are associated with insulin signal transduction, were assessed in the basal or the insulin­stimulated state using western blot analysis or RT-PCR. We also examined the mRNA expression of monocyte chemotactic protein 1 (MCP-1), interleukin 6 (IL-6), tumor necrosis factor α (TNFα) and Toll­like receptor 4 (TLR4). Taken together, these findings demonstrated that astragaloside IV facilitates glucose transport in C2C12 myotubes through a mechanism involving the IRS1/AKT pathway, and suppresses the palmitate-induced activation of the IKK/IκBα pathway.

Synthesis of chalcone derivatives as potential anti-diabetic agents.

Chalcones bearing electron donating or electron withdrawing substitutions were prepared and their glucose uptake activity was evaluated. Chalcone derivatives were synthesized in one step protocol with high purity and yield. Chalcones with chloro, bromo, iodo and hydroxy substitutions at position 2 on A-ring exhibited the highest activity with glucose medium concentration (210 to 236 mg/dl) compared to pioglitazone and rosiglitazone (230 and 263 mg/dl, respectively). Also chalcones with iodo substitution at position 3 on A-ring were comparably active (≤238 mg/dl). The structure-activity relationship of the tested chalcones was studied and the findings were supported statistically.

Interleukin-6 released from fibroblasts is essential for up-regulation of matrix metalloproteinase-1 expression by U937 macrophages in coculture: cross-talking between fibroblasts and U937 macrophages exposed to high glucose.

Matrix metalloproteinases (MMPs) play a key role in periodontal disease. Although it is known that macrophages and fibroblasts are co-localized and express MMPs in the diseased periodontal tissue, the effect of interaction between these two cell types on MMP expression has not been well elucidated. Furthermore although it is known that diabetes is associated with accelerated periodontal tissue destruction, it remains unknown whether hyperglycemia, a major metabolic abnormality in diabetes, regulates MMP expression by affecting the cross-talking between fibroblasts and macrophages. In this study, human gingival fibroblasts and U937 macrophages were cocultured in a two-compartment transwell culture system, and the cells were treated with normal or high glucose. We found that coculture of fibroblasts and U937 macrophages led to an augmentation of MMP-1 expression by U937 macrophages, and high glucose further enhanced this augmentation. Similar observations were also made in the coculture of fibroblasts and human primary monocytes. We also found that interleukin 6 (IL-6) released by fibroblasts was essential for the augmentation of MMP-1 expression by U937 macrophages. Furthermore our results showed that high glucose, IL-6, and lipopolysaccharide had a synergistic effect on MMP-1 expression. Finally our study indicated that MAPK pathways and activator protein-1 transcription factor were involved in the coculture- and high glucose-augmented MMP-1 expression. In conclusion, this study demonstrates that IL-6 derived from fibroblasts is essential for MMP-1 up-regulation by cross-talking between fibroblasts and U937 macrophages exposed to high glucose, revealing an IL-6-dependent mechanism in MMP-1 up-regulation.

Oleanolic acid derivative NPLC441 potently stimulates glucose transport in 3T3-L1 adipocytes via a multi-target mechanism.

The natural product oleanolic acid (OA) has been discovered to exhibit varied pharmacological functions including anti-inflammation, anti-tumor and anti-diabetes, while appropriate synthetic oleanolic acid derivatives seem to possess more potent activities. Here we identified a new oleanolic acid derivative, 3-beta-(2-carboxybenzoyloxy)-oleanolic acid (NPLC441), which functioned as a competitive PTP1B inhibitor and enhanced insulin-stimulated phosphorylation of IR and AKT in HepG2 cells. As an RXRalpha antagonist, it could selectively activate LXRalpha:RXRalpha heterodimer and increase the promoter activities of ABCA1 and ABCG1 genes in transient transfection assays. Quantitative RT-PCR and Western blot analyses suggested that NPLC441 could up-regulate GLUT4 expression in 3T3-L1 adipocytes, and such effect was further proved to be dependent on LXRalpha:RXRalpha activation. Moreover, 2-deoxyglucose uptake technology-based characterization demonstrated that this compound could stimulate glucose uptake in 3T3-L1 adipocytes. Finally, NPLC441 was observed to be able to suppress 11beta-HSD(1) expression in HepG2 cells, following the discovery that activation of LXRalpha:RXRalpha could repress the expression of 11beta-HSD(1). Compared with NPLC441, OA showed no effects on the transactivation of either LXRalpha:RXRalpha heterodimer or RXRalpha-LBD. Our work is thus expected to provide a new insight into the anti-diabetic application for oleanolic acid derivatives via multi-target mechanism, and NPLC441 could be used as a potential lead compound for further research.

Acute regulation of glucose transport in a human megakaryocytic cell line: difference between growth factors and H(2)O(2).

The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H(2)O(2) on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H(2)O(2); (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H(2)O(2): cytokines increased predominantly the affinity for glucose, while H(2)O(2) raised both the V(max) and K(m) values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H(2)O(2)-induced stimulation of hexose transport, reversing the variations of K(m) and V(max), but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H(2)O(2) on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor.

Effects of S-nitroso-N-acetyl-penicillamine administration on glucose tolerance and plasma levels of insulin and glucagon in the dog.

It has been suggested that nitric oxide (NO, nitrogen monoxide) is a regulator of carbohydrate metabolism in skeletal muscle. The present study was undertaken to investigate the acute effects of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on blood glucose levels and on the gluco-regulatory hormones insulin and glucagon in healthy dogs. The acute effects of SNAP on mean arterial pressure and heart rate were also investigated. The drug was administered intravenously and the pre- and postprandial blood glucose, plasma insulin, and glucagon concentrations were determined at half-hour time intervals postadministration after a glucose challenge. The plasma nitrate and nitrite concentrations were measured and taken as the biochemical markers of in vivo NO formation. The oral glucose tolerance test revealed an impaired glucose tolerance in SNAP-treated dogs as reflected by the area under the glucose curve, 1150.50 +/- 63.00 mmol x 150 min and 1355.25 +/- 102.01 mmol/L x 150 min in dogs treated with 10 and 20 mg/kg of SNAP, respectively, compared with 860.25 +/- 60.68 mmol/L x 150 min in captopril-treated controls (P < 0.05). The 2-h blood glucose concentration in dogs treated with 20 mg/kg body wt of SNAP was 9.17 +/- 1.10 mmol/L compared with 5.59 +/- 0.26 mmol/L for captopril-treated controls (P = 0.015). The oral glucose tolerance test also confirmed an impaired insulin secretion in the SNAP-treated dogs. While the plasma insulin concentration increased gradually in the captopril-treated controls to a peak value of 39.50 +/- 2.55 microIU/ml, 1.5 h after a glucose challenge there was a decrease in the plasma insulin concentration in SNAP-treated dogs to a low value of 20.67 +/- 0.88 microIU/ml (P = 0.006). In contrast, there were no significant differences in plasma glucagon concentration in SNAP-treated dogs and captopril-treated dogs at any time points. Using the Griess reaction, we found that there was a 27-95% increase in plasma nitrate/nitrite concentration on administration of SNAP. The sustained hyperglycemic effect observed in SNAP-treated dogs was accompanied by a marginal decrease in the mean arterial blood pressure and a significant increase in heart rate (P < 0.05). We conclude that acute administration of SNAP in the oral glucose tolerance test releases NO that modulates the parameters of carbohydrate metabolism.

Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic beta-cells.

Glucose gradients generated by an artificial source and beta-cells were measured using an enzyme-based glucose microsensor, 8-microm tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 +/- 0.4 pmol.cm(-2).s(-1) (mean +/- SEM, n = 7) with a sensor sensitivity of 7.0 +/- 0.4 pA/ mM (mean +/- SEM, n = 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from beta-cell clusters, measured in the presence of 5 mM glucose, was 61.7 +/- 9.5 fmol.nL(-1).s(-1) (mean +/- SEM, n = 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 microM) transiently increased, subsequently decreasing to below basal by 93 +/- 16 and 56 +/- 6%, respectively (mean +/- SEM, n = 5). Consumption was decreased after the application of 10 microM rotenone by 74 +/- 5% (mean +/- SEM, n = 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment.

Pretreatment with glucose increases entry of urocortin into mouse brain.

Although urocortin is a potent inhibitor of food ingestion after peripheral administration, it was recently shown that under normal conditions this peptide crosses the blood-brain barrier (BBB) at a very slow rate. We examined whether hyperglycemia could stimulate the rate of entry (K(i)) of (125)I-urocortin into the mouse brain. In euglycemic mice, (125)I-urocortin injected iv entered the brain at a rate similar to that of the vascular marker (99m)Tc-albumin. However, injection of glucose (3 g/kg, ip) 0.5, 1, or 2 h before the (125)I-urocortin greatly increased the influx of urocortin. Without the glucose, the self-inhibition characteristic of a saturable transport system was not apparent. Self-inhibition could be demonstrated after the glucose injection, indicating activation of a transport system for urocortin that was saturable. Injection of insulin (10 U/kg, ip) 1 or 2 h before the (125)I-urocortin decreased the K(i). Thus, the entry of urocortin into brain can be activated by changes in the concentration of blood glucose, illustrating the responsiveness of the BBB to regulatory influences.

Isomeric and anomeric specificity of the metabolic and secretory response of rat pancreatic islets to glucose pentaacetate.

In the presence of 2.8 mM D-glucose, beta-D-glucose pentaacetate (1. 7 mM) augmented insulin release from isolated rat pancreatic islets more than alpha-D-glucose pentaacetate. Likewise, the further increment in insulin output evoked by nateglinide (0.01 mM) was higher in islets exposed to beta- rather than alpha-D-glucose pentaacetate. Inversely, in the presence of 2.8 mM unesterified D-glucose, alpha-L-glucose pentaacetate, but not beta-L-glucose pentaacetate, significantly augmented insulin output. The higher insulinotropic potency of the beta-anomer of D-glucose pentaacetate coincided with the fact that it significantly increased the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, whereas alpha-D-glucose pentaacetate failed to do so. These findings are interpreted to support the concept that the stimulation of insulin release by these esters is largely attributable to their direct interaction with a stereospecific receptor, with preference for the configuration of the C1 common to beta-D-glucose pentaacetate and alpha-L-glucose pentaacetate.