Absence Of Glucose In Medium Research Articles

The Transcriptional Response of Candida albicans to Weak Organic Acids, Carbon Source, and MIG1 Inactivation Unveils a Role for HGT16 in Mediating the Fungistatic Effect of Acetic Acid.

Candida albicans is a resident fungus of the human intestinal microflora. Commonly isolated at low abundance in healthy people, C. albicans outcompetes local microbiota during candidiasis episodes. Under normal conditions, members of the human gastrointestinal (GI) microbiota were shown to keep C. albicans colonization under control. By releasing weak organic acids (WOAs), bacteria are able to moderate yeast growth. This mechanism displays a synergistic effect in vitro with the absence of glucose in medium of culture, which underlines the complex interactions that C. albicans faces in its natural environment. Inactivation of the transcriptional regulator MIG1 in C. albicans results in a lack of sensitivity to this synergistic outcome. To decipher C. albicans transcriptional responses to glucose, WOAs, and the role of MIG1, we performed RNA sequencing (RNA-seq) on four biological replicates exposed to combinations of these three parameters. We were able to characterize the (i) glucose response, (ii) response to acetic and butyric acid, (iii) MIG1 regulation of C. albicans, and (iv) genes responsible for WOA resistance. We identified a group of six genes linked to WOA sensitivity in a glucose-MIG1-dependent manner and inactivated one of these genes, the putative glucose transporter HGT16, in a SC5314 wild-type background. As expected, the mutant displayed a partial complementation to WOA resistance in the absence of glucose. This result points toward a mechanism of WOA sensitivity in C. albicans involving membrane transporters, which could be exploited to control yeast colonization in human body niches.

Geranylgeranylacetone ameliorates ischemic acute renal failure via induction of Hsp70

Heat shock proteins (HSPs) are well known as cytoprotective proteins. Geranylgeranylacetone (GGA), an antiulcer agent, has recently been shown to induce Hsp70. This study was performed to investigate the renoprotective properties of GGA. The effect of GGA on the induction of the major HSPs (Hsp90, Hsp70, Hsc70, Hsp60, and Hsp32) was studied in the rat kidney or rat primary cultures of tubular epithelial cells (R-TECs) by Western blot. Localization of Hsp70 was determined by immunohistochemistry. The renoprotective effects of GGA were studied using a rat model of ischemia/reperfusion (I/R) injury. GGA (400 mg/kg), GGA with quercetin pretreatment (100 mg/kg), or a vehicle was given to rats 24 hours and again 1 hour prior to the induction of I/R injury. Rats were sacrificed at 24 hours after reperfusion. Histologic analyses and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay were performed. Blood urea nitrogen (BUN) and serum creatinine was also measured. The cytoprotective properties of GGA were also studied in vitro by treating R-TECs with GGA (10 mumol/L) or a vehicle, followed by incubation in culture medium with oxidative stress condition (0.5 mmol/L hydrogen peroxide) or ischemic condition (2 nmol/L NaCN and 20 mmol/L 2-deoxyglucose in the absence of medium glucose). Oral administration of GGA induced Hsp70 expression in the kidney (which peaked at 24 hours) but did not induce Hsp90, Hsc70, Hsp60, or Hsp32. The induction of Hsp70 was blocked by quercetin. Immunohistochemistry showed that Hsp70 was localized mainly in the tubular epithelial cells. Preconditioning rats with GGA significantly decreased BUN and serum creatinine levels after I/R injury. Histologic examination revealed that GGA significantly attenuated tubular damage and macrophage infiltration. The number of TUNEL-positive cells also decreased significantly in the GGA group. Quercetin, an inhibitor of Hsp70 induction, eliminated these renoprotective effects of GGA. In in vitro study, GGA-induced Hsp70 in R-TECs, which peaked at 2 to 4 hours. Both oxidative stress and ischemic stimuli induced apoptosis in R-TECs. GGA significantly suppressed the number of apoptotic cells in both conditions. The results support the hypothesis that GGA induces Hsp70, protects tubular epithelial cells from apoptosis, and thus ameliorates tubular damage by I/R injury. The present study suggests that GGA would be a useful tool in treating acute renal failure or preventing transplanted kidney damage in the clinical setting.

HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells.

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an F0F1 ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner (r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation.

Hsp72 Inhibits Focal Adhesion Kinase Degradation in ATP-depleted Renal Epithelial Cells

Prior heat stress (HS) or the selective overexpression of hsp72 prevents apoptosis caused by exposure to metabolic inhibitors by protecting the mitochondrial membrane and partially reducing caspase-3 activation. Focal adhesion kinase (FAK), a tyrosine kinase, exhibits anti-apoptotic properties and is a potential target for degradation by caspase-3. This study tested the hypothesis that hsp72 interacts with FAK, preventing caspase-3-mediated degradation during ATP depletion. ATP depletion (5 mm NaCN and 5 mm 2-deoxy-d-glucose in the absence of medium glucose) caused FAK degradation within 15 min. FAK degradation was completely prevented by a caspase-3-specific inhibitor. HS induced the accumulation of hsp72, increased the interaction between hsp72 and FAK, and significantly inhibited FAK degradation during ATP depletion. Selective overexpression of wild-type hsp72 (but not hsp72DeltaEEVD) reproduced the protective effects of HS on FAK cleavage. Purified hsp72 prevented the degradation of FAK by caspase-3 in vitro in a dose-dependent manner without affecting caspase-3 activity. Interaction between hsp72 and FAK is critical because both exogenous ATP and deletion of the substrate-binding site decreased protection of FAK by hsp72. These data indicate that FAK is an early target of injury in cells exposed to metabolic inhibitors and demonstrate that hsp72 reduces caspase-3-mediated proteolysis of FAK, an anti-apoptotic protein.

Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro.

The effect of glucose in the medium used during in vitro culture on the sex ratio of bovine blastocysts derived from in-vitro-matured and in-vitro-fertilized oocytes was evaluated. Oocytes were matured and inseminated with mixed sperm from three bulls and were cultured in vitro in modified synthetic oviducal fluid medium with 10% fetal calf serum, with or without glucose supplementation. The overall rate of cleaved embryos that developed to expanded blastocyst in the medium without glucose (27.0%) was significantly greater (P < 0.05) than the percentage observed when embryos were cultured in medium with glucose (17.5%). Analysis of variance was performed to analyse the effect of glucose on the proportion of male embryos reaching the blastocyst stage (or arrested at the morula stage) during Days 7 to 10. Regardless of the presence or absence of glucose in the medium, significantly (P < 0.05) more male than female embryos were harvested as expanded blastocysts on Day 7 and on Day 8 of culture. On Days 9 plus 10 of culture, a sex ratio imbalance only occurred in the absence of glucose in the culture medium (P < 0.05). Glucose did not produce any significant effect on the sex ratio of the overall number of expanded blastocysts harvested by Day 10 of in vitro culture. However a significantly greater proportion of females (P < 0.01) were found among those embryos that developed only to the morulae stage after 10 days in vitro. These results show that glucose supplementation of culture media produces a preferential loss of female embryos during culture to the blastocyst stage.

Prior heat stress enhances survival of renal epithelial cells after ATP depletion.

The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with the steady-state content of HSP-72. In the recovery period after ATP depletion, cell ATP content, maximum mitochondrial ATP production rate, and total LDH activity were all significantly higher in cells with abundant HSP-72. Although the protective effects associated with heat stress are likely to be multifactoral, preserved cell metabolism and higher ATP content could enhance cellular repair processes after ATP depletion.

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.

Local Response of Muscle to Burns: Relationship of Glycolysis and Amino Acid Release

The study examined the cause of, and relationship between, the increased rates of glucose utilization and amino acid release that are uniquely exhibited by skeletal muscle underlying the burn wound. Ether‐anesthetized rats were subjected to a 3‐sec one hind limb‐scald and 3 days later glucose and amino acid metabolism of soleus muscles from the burned and unburned limbs of burned rats as well as from controls was studied in vitro. Metabolism of the unburned‐limb muscle did not differ from control under any experimental conditions. In contrast, muscles from the burned limb took up to 3 times more glucose than controls in 10 mM glucose. Anoxia increased glucose uptake by all muscle groups but did not eliminate the relative enhancement exhibited by the burned‐limb muscles. Muscles from the burned limb also released 101% more alanine, 68% more glutamate, 110% more glutamine, and 163% more tyrosine in the presence or absence of medium glucose. With the exception of a uniform increase in glutamate release, anoxia did not alter amino acid releases by any muscle group. Inclusion of 1 mM iodoacetate in the glucose‐free medium reduced alanine release and raised glutamate release in all muscle groups, eliminating their relatively enhanced release by burned‐limb muscles. Iodoacetate markedly diminished glutamine release by muscles from the burned limb and caused a relatively depressed glutamine release by these muscles as compared to control. The fact that ATP‐MgCl2 reversed the depressed release of glutamine by burned‐limb muscles suggests that cellular ATP is the limiting factor for glutamine synthesis by these muscles in iodoacetate. It is concluded that muscle hypoxia is only partially responsible for the increased glucose utilization by muscle from the burned region, and does not contribute to the augmented release of amino acids. The stimulated alanine release by muscle from the burned region does, however, appear to depend upon the increased pyruvate supply evident in these muscles.

Stimulation of Glycolytic Activity of Rat Corpus Luteum Tissue by Luteinizing Hormone

Investigations of the glucose metabolism of rat luteinized ovary tissue have been undertaken in attempts to shed further light upon the stimulatory action of luteinizing hormone on this tissue. Studies with glucose-U-C14 in vitro indicate that respiratory CO2, lipids and lactic acid on the average account for 13, 2 and 74%, respectively, of the amount of glucose taken up by the tissue. These percentages remain essentially unaltered when the rate of glucose uptake is increased by LH treatment in vivo. Rate of lactic acid formation increases with increasing medium glucose concentration, essentially reaching a plateau at 16 mg glucose/ ml. LH treatment in vivo results in stimulation of lactic acid formation at all medium glucose concentrations tested. Lactic acid formation from endogenous materials in the absence of medium glucose is very small, a finding consistent with negligible amounts of glycogen found in rat luteal tissue. The ability of LH to stimulate lactic acid production and glucose uptake is grea...