Voluntary Behavior and Training Conditions Modulate in vivo Extracellular Glucose and Lactate in the Mouse Primary Motor Cortex

Learning or performing new behaviors requires significant neuronal signaling and is metabolically demanding. The metabolic cost of performing a behavior is mitigated by exposure and practice which result in diminished signaling and metabolic requirements. We examined the impact of novel and habituated wheel running, as well as effortful behaviors on the modulation of extracellular glucose and lactate using biosensors inserted in the primary motor cortex of mice. We found that motor behaviors produce increases in extracellular lactate and decreases in extracellular glucose in the primary motor cortex. These effects were modulated by experience, novelty and intensity of the behavior. The increase in extracellular lactate appears to be strongly associated with novelty of a behavior as well as the difficulty of performing a behavior. Our observations are consistent with the view that a main function of aerobic glycolysis is not to fuel the current neuronal activity but to sustain new bio-infrastructure as learning changes neural networks, chiefly through the shuttling of glucose derived carbons into the pentose phosphate pathway for the biosynthesis of nucleotides.

The Role of TRAPγ/SSR3 in Preproinsulin Translocation into the Endoplasmic Reticulum

In the endoplasmic reticulum (ER), the Translocation-Associated Protein complex (TRAP, also called Signal sequence receptor, SSR) includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2 and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2 or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting co-translational and post-translational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels — observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkable, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

The Role of TRAPγ/SSR3 in Preproinsulin Translocation into the Endoplasmic Reticulum

In the endoplasmic reticulum (ER), the Translocation-Associated Protein complex (TRAP, also called Signal sequence receptor, SSR) includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2 and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2 or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting co-translational and post-translational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels — observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkable, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

Analysis of the Glucose-Dependent Transcriptome in Murine Hypothalamic Cells

We have previously shown that glucose activates CREB-regulated transcriptional co-activator-2 (CRTC-2) in murine, hypothalamic (mHypoA-2/10) cells. Thus, we now analysed the entire glucose-dependent transcriptome of mHypoA-2/10 cells by total RNA-seq. 831 genes were up- and 1390 genes down-regulated by at least 50 %. Signalling pathway analysis revealed activation of the cholesterol biosynthesis pathway by glucose. Accordingly, protein expression of both sterol regulatory element-binding proteins (SREBP) and total cholesterol levels were enhanced by glucose. Analysis of single genes involved in fundamental signalling processes suggested a significant impact of glucose. Thus, we chose ~100 genes and validated the effects of glucose on mRNA levels by qRT-PCR. We identified 15 genes with strong glucose-dependent mRNA expression. Among these genes were gnai1 to -3, adyc6, irs1, igfr1, hras and elk3. cAMP measurements revealed decreased basal and enhanced noradrenalin-induced cAMP levels at higher glucose concentrations. Serum-response element-dependent reporter assays indicated enhanced basal and insulin-like growth factor-induced activity at higher glucose levels. siRNA against CRTC-2 dampened the effects of glucose on cholesterol synthesis and IRS-1, SREBP-1, SREBP-2 or AC-6 protein expression. These findings could help to understand the functional consequences of physiologically occurring alterations of extracellular glucose concentrations as well as pathologically increased glucose levels.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Short Note: Extracellular Export and Consumption of Glucose in Antarctic Sea Ice

Glucose microsensors were used to measure extracellular glucose exudation and consumption in a sea ice algal community. Glucose export increased with increasing irradiance between 15 and 512 µmol photons m− 2 s− 1. This export correlated with declining FvFm values and increasing NPQ values, implying that glucose export resulted from exposure to above optimal irradiances. Glucose concentrations in samples treated with DCMU to block photosynthesis, declined at all irradiances. Bacterial consumption of glucose was between 6% and 34% of extracellular export per hour.

Regulation of Fructose 1,6-Bisphosphatase in Procyclic Form Trypanosoma brucei

Glycolysis is well described in Trypanosoma brucei, while the importance of gluconeogenesis and one of the key enzymes in that pathway, fructose 1,6-bisphosphatase, is less understood. Using a sensitive and specific assay for FBPase, we demonstrate that FBPase activity in insect stage, procyclic form (PF), parasite changes with parasite cell line, extracellular glucose levels, and cell density. FBPase activity in log phase PF 2913 cells was highest in high glucose conditions, where gluconeogenesis is expected to be inactive, and was undetectable in low glucose, where gluconeogenesis is predicted to be active. This unexpected relationship between FBPase activity and extracellular glucose levels suggests that FBPase may not be exclusively involved in gluconeogenesis and may play an additional role in parasite metabolism. In stationary phase cells, the relationship between FBPase activity and extracellular glucose levels was reversed. Furthermore, we found that monomorphic PF 2913 cells had significantly higher FBPase levels than pleomorphic PF AnTat1.1 cells where the activity was undetectable except when cells were grown in standard SDM79 media, which is glucose-rich and commonly used to grow PF trypanosomes in vitro. Finally, we observed several conditions where FBPase activity changed while protein levels did not, suggesting that the enzyme may be regulated via post-translational modifications.