Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in Bacillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Local osmotic gradients drive the water flux associated with Na+/glucose cotransport

It recently was proposed [Loo, D. D. F., Zeuthen, T., Chandy, G. & Wright, E. M. (1996) Proc. Natl. Acad. Sci. USA 93, 13367–13370] that SGLT1, the high affinity intestinal and renal sodium/glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na+, one glucose, and ≈220 water molecules per transport cycle. Using electrophysiology together with sensitive volumetric measurements, we investigated the nature of the driving force behind the cotransporter-mediated water flux. The osmotic water permeability of oocytes expressing human SGLT1 (Lp ± SE) averaged 3.8 ± 0.3 × 10−4 cm⋅s−1 (n = 15) and addition of 100 μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ± 0.2 × 10−4 cm⋅s−1 (n = 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM α-methyl-glucose (αMG) induced an average inward current of 800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL cm−2 ⋅s−1 within 5–8 min. After rapidly inhibiting the Na+/glucose cotransport with phlorizin, the water flux remained significantly elevated, clearly indicating the presence of a local osmotic gradient (Δπ) estimated at 16 ± 2 mOsm. In short-term experiments, a rapid depolarization from −100 to 0 mV in the presence of αMG decreased the cotransport current by 94% but failed to produce a comparable reduction in the swelling rate. A mathematical model depicting the intracellular accumulation of transported osmolytes can accurately account for these observations. It is concluded that, in SGLT1-expressing oocytes, αMG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Mannose Inhibits Arabidopsis Germination via a Hexokinase-Mediated Step1

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3− assimilation by C. reinhardtii.

Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.

Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose. We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose. This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p. We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose. This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes. Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene VI coding sequence was placed under the control of the galactose-inducible promoter GAL1, which is presented in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL1 promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

The Effect of Fructose vs. Glucose Beverages on Low-Grade Systemic Inflammation

Thesis (Ph.D.)--University of Washington, 2016-04

High glucose transactivates the EGF receptor and up-regulates serum glucocorticoid kinase in the proximal tubule

Background. Serum glucocorticoid regulated kinase (SGK-1) is induced in the kidney in diabetes mellitus. However, its role in the proximal tubule is unclear. This study determined the expression and functional role of SGK-1 in PTCs in high glucose conditions. As the epidermal growth factor (EGF) receptor is activated by both EGF and other factors implicated in diabetic nephropathy, the relationship of SGK-1 with EGFR activity was assessed. Methods. mRNA and protein expression of SGK-1 and mRNA expression of the sodium hydrogen exchanger NHE3 were measured in human PTCs exposed to 5 mmol/L (control) and 25 mmol/L (high) glucose. The effects of SGK-1 on cell growth, apoptosis, and progression through the cell cycle and NHE3 mRNA were examined following overexpression of SGK-1 in PTCs. The role of EGFR activation in observed changes was assessed by phospho-EGFR expression, and response to the EGFR blocker PKI166. SGK-1 expression was then assessed in vivo in a model of streptozotocin-induced diabetes mellitus type 2. Results. A total of 25 mmol/L glucose and EGF (10 ng/mL) increased SGK-1 mRNA (P < 0.005 and P < 0.002, respectively) and protein (both P < 0.02) expression. High glucose and overexpression of SGK-1 increased NHE3 mRNA (P < 0.05) and EGFR phosphorylation (P < 0.01), which were reversed by PKI166. SGK-1 overexpression increased PTC growth (P < 0.0001), progression through the cell cycle (P < 0.001), and increased NHE3 mRNA (P < 0.01), which were all reversed with PKI166. Overexpression of SGK-1 also protected against apoptosis induced in the PTCs (P < 0.0001). Up-regulation of tubular SGK-1 mRNA in diabetes mellitus was confirmed in vivo. Oral treatment with PKI166 attenuated this increase by 51%. No EGF protein was detectable in PTCs, suggestive of phosphorylation of the EGFR by high glucose and downstream induction of SGK-1. Conclusion. The effects of high glucose on PTC proliferation, reduced apoptosis and increased NHE3 mRNA levels are mediated by EGFR-dependent up-regulation of SGK-1.

Carotid artery intimal medial thickness, brachial artery flow-mediated vasodilation and cardiovascular risk factors in diabetic and non-diabetic indigenous Australians

Background: Indigenous Australians are at high risk for cardiovascular disease and type 2 diabetes. Carotid artery intimal medial thickness (CIMT) and brachial artery flow-mediated vasodilation (FMD) are ultrasound imaging based surrogate markers of cardiovascular risk. This study examines the relative contributions of traditional cardiovascular risk factors on CIMT and FMD in adult Indigenous Australians with and without type 2 diabetes mellitus. Method: One hundred and nineteen Indigenous Australians were recruited. Physical and biochemical markers of cardiovascular risk, together with CIMT and FMD were meausred for all subjects. Results: Fifty-three Indigenous Australians subjects (45%) had type 2 diabetes mellitus. There was a significantly greater mean CIMT in diabetic versus non-diabetic subjects (p = 0.049). In the non-diabetic group with non-parametric analyses, there were significant correlations between CIMT and: age (r = 0.64, p < 0.001), systolic blood pressure (r = 0.47, p < 0.001) and non-smokers (r = -0.30, p = 0.018). In the diabetic group, non-parametric analysis showed correlations between CIMT, age (r = 0.36, p = 0.009) and duration of diabetes (r = 0.30, p = 0.035) only. Adjusting forage, sex, smoking and history of cardiovascular disease, Hb(A1c) became the sole significant correlate of CIMT (r = 0.35,p = 0.01) in the diabetic group. In non-parametric analysis, age was the sole significant correlate of FMD (r = -0.31,p = 0.013), and only in non-diabetic subjects. Linear regression analysis showed significant associations between CIMT and age (t = 4.6,p < 0.001), systolic blood pressure (t = 2.6, p = 0.010) and Hb(A1c) (t = 2.6, p = 0.012), smoking (t = 2.1, p = 0.04) and fasting LDL-cholesterol (t = 2.1, p = 0.04). There were no significant associations between FMD and examined cardiovascular risk factors with linear regression analysis Conclusions: CIMT appears to be a useful surrogate marker of cardiovascular risk in this sample of Indigenous Australian subjects, correlating better than FMD with established cardiovascular risk factors. A lifestyle intervention programme may alleviate the burden of cardiovascular disease in Indigenous Australians by reducing central obesity, lowering blood pressure, correcting dyslipidaemia and improving glycaemic control. CIMT may prove to be a useful tool to assess efficacy of such an intervention programme. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro:implications for pancreatic islet transplantation

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p <0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of ß-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.