Repetitive Short-Term Stimuli Imposed in Poor Mixing Zones Induce Long-Term Adaptation of E. coli Cultures in Large-Scale Bioreactors: Experimental Evidence and Mathematical Model.

Rapidly changing concentrations of substrates frequently occur during large-scale microbial cultivations. These changing conditions, caused by large mixing times, result in a heterogeneous population distribution. Here, we present a powerful and efficient modeling approach to predict the influence of varying substrate levels on the transcriptional and translational response of the cell. This approach consists of two parts, a single-cell model to describe transcription and translation for an exemplary operon (trp operon) and a second part to characterize cell distribution during the experimental setup. Combination of both models enables prediction of transcriptional patterns for the whole population. In summary, the resulting model is not only able to anticipate the experimentally observed short-term and long-term transcriptional response, it further allows envision of altered protein levels. Our model shows that locally induced stress responses propagate throughout the bioreactor, resulting in temporal, and spatial population heterogeneity. Stress induced transcriptional response leads to a new population steady-state shortly after imposing fluctuating substrate conditions. In contrast, the protein levels take more than 10 h to achieve steady-state conditions.

Transcriptional response of Escherichia coli to ammonia and glucose fluctuations.

In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients such as glucose or ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programmes. Previously, we characterized short- and long-term strategies of cells to adapt to glucose fluctuations. Here, we focused on fluctuating ammonia supply while studying a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug-flow reactor (PFR). The alarmone ppGpp rapidly accumulated in PFR, initiating considerable transcriptional responses after 70 s. About 400 genes were repeatedly switched on/off when E. coli returned to the STR. E. coli revealed highly diverging long-term transcriptional responses in ammonia compared to glucose fluctuations. In contrast, the induction of stringent regulation was a common feature of both short-term responses. Cellular ATP demands for coping with fluctuating ammonia supply were found to increase maintenance by 15%. The identification of genes contributing to the increased ATP demand together with the elucidation of regulatory mechanisms may help to create robust cells and processes for large-scale application.

Switching between nitrogen and glucose limitation: Unraveling transcriptional dynamics in Escherichia coli.

Transcriptional control under nitrogen and carbon-limitation conditions have been well analyzed for Escherichia coli. However, the transcriptional dynamics that underlie the shift in regulatory programs from nitrogen to carbon limitation is not well studied. In the present study, cells were cultivated at steady state under nitrogen (ammonia)-limited conditions then shifted to carbon (glucose) limitation to monitor changes in transcriptional dynamics. Nitrogen limitation was found to be dominated by sigma 54 (RpoN) and sigma 38 (RpoS), whereas the "housekeeping" sigma factor 70 (RpoD) and sigma 38 regulate cellular status under glucose limitation. During the transition, nitrogen-mediated control was rapidly redeemed and mRNAs that encode active uptake systems, such as ptsG and manXYZ, were quickly amplified. Next, genes encoding facilitators such as lamB were overexpressed, followed by high affinity uptake systems such as mglABC and non-specific porins such as ompF. These regulatory programs are complex and require well-equilibrated and superior control. At the metabolome level, 2-oxoglutarate is the likely component that links carbon- and nitrogen-mediated regulation by interacting with major regulatory elements. In the case of dual glucose and ammonia limitation, sigma 24 (RpoE) appears to play a key role in orchestrating these complex regulatory networks.

Engineering E. coli for large-scale production - Strategies considering ATP expenses and transcriptional responses.

Microbial producers such as Escherichia coli are evolutionarily trained to adapt to changing substrate availabilities. Being exposed to large-scale production conditions, their complex, multilayered regulatory programs are frequently activated because they face changing substrate supply due to limited mixing. Here, we show that E. coli can adopt both short- and long-term strategies to withstand these stress conditions. Experiments in which glucose availability was changed over a short time scale were performed in a two-compartment bioreactor system. Quick metabolic responses were observed during the first 30s of glucose shortage, and after 70s, fundamental transcriptional programs were initiated. Since cells are fluctuating under simulated large-scale conditions, this scenario represents a continuous on/off switching of about 600 genes. Furthermore, the resulting ATP maintenance demands were increased by about 40-50%, allowing us to conclude that hyper-producing strains could become ATP-limited under large-scale production conditions. Based on the observed transcriptional patterns, we identified a number of candidate gene deletions that may reduce unwanted ATP losses. In summary, we present a theoretical framework that provides biological targets that could be used to engineer novel E. coli strains such that large-scale performance equals laboratory-scale expectations.

Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle.

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.

Phosphate limited fed-batch processes: impact on carbon usage and energy metabolism in Escherichia coli.

Phosphate starvation is often applied as a tool to limit cell growth in microbial production processes without hampering carbon and/or nitrogen supply alternatively. This contribution focuses on the interplay of process induced phosphate starvation and microbial performance studying an l-tryptophan overproducing Escherichia coli strain as a model for highly ATP demanding processes in comparison with an E. coli wildtype strain. To enable a time-resolved analysis, constant phosphate feeding strategies were applied to elongate the transition from phosphate saturated to phosphate limited cell growth. With increasing phosphate limitation, a reduced cellular efficiency of ATP formation via respiratory chain activity and the ATP synthase complex was found for both strains. Process balancing, transcriptome analysis and flux balance analysis are pointing toward a multi-stage decoupling scenario, which in essence deteriorates the stoichiometric ratio of ATP formation to proton translocation, thereby affecting ATP availability from respiration and carbon usage. Starting off with a potential influence on ATP-synthase efficiency (stage 1), decoupling is further increased by modified respiratory activity (stage 2) and byproduct overflow (stage 3) finally resulting in a metabolic breakdown entering complete phosphate depletion (stage 4). The decoupling is initiated by phosphate limitation; further effects are mainly mediated on metabolic level through ATP availability and energy charge, additionally affected by ATP demanding product synthesis.

Enhanced fasting glucose turnover in mice with disrupted action of TUG protein in skeletal muscle.

Insulin stimulates glucose uptake in 3T3-L1 adipocytes in part by causing endoproteolytic cleavage of TUG (tether containing a ubiquitin regulatory X (UBX) domain for glucose transporter 4 (GLUT4)). Cleavage liberates intracellularly sequestered GLUT4 glucose transporters for translocation to the cell surface. To test the role of this regulation in muscle, we used mice with muscle-specific transgenic expression of a truncated TUG fragment, UBX-Cter. This fragment causes GLUT4 translocation in unstimulated 3T3-L1 adipocytes. We predicted that transgenic mice would have GLUT4 translocation in muscle during fasting. UBX-Cter expression caused depletion of PIST (PDZ domain protein interacting specifically with TC10), which transmits an insulin signal to TUG. Whereas insulin stimulated TUG proteolysis in control muscles, proteolysis was constitutive in transgenic muscles. Fasting transgenic mice had decreased plasma glucose and insulin concentrations compared with controls. Whole-body glucose turnover was increased during fasting but not during hyperinsulinemic clamp studies. In muscles with the greatest UBX-Cter expression, 2-deoxyglucose uptake during fasting was similar to that in control muscles during hyperinsulinemic clamp studies. Fasting transgenic mice had increased muscle glycogen, and GLUT4 targeting to T-tubule fractions was increased 5.7-fold. Whole-body oxygen consumption (VO2), carbon dioxide production (VCO2), and energy expenditure were increased by 12-13%. After 3 weeks on a high fat diet, the decreased fasting plasma glucose in transgenic mice compared with controls was more marked, and increased glucose turnover was not observed; the transgenic mice continued to have an increased metabolic rate. We conclude that insulin stimulates TUG proteolysis to translocate GLUT4 in muscle, that this pathway impacts systemic glucose homeostasis and energy metabolism, and that the effects of activating this pathway are maintained during high fat diet-induced insulin resistance in mice.

Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

The role of peroxisome proliferator-activated receptor gamma coactivator-1 beta in the pathogenesis of fructose-induced insulin resistance.

Peroxisome proliferator-activated receptor gamma coactivator-1 beta (PGC-1beta) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here, we evaluated the role of PGC-1beta in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1beta in liver and adipose tissue. PGC-1beta ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1beta ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin-stimulated states. Furthermore, PGC-1beta ASO increased insulin-stimulated whole-body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1beta in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1beta inhibition may be a therapeutic target for treatment of NAFLD, hypertriglyceridemia, and insulin resistance associated with increased de novo lipogenesis.

The glucose transporter 4-regulating protein TUG is essential for highly insulin-responsive glucose uptake in 3T3-L1 adipocytes.

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.

Blood glucose-lowering nuclear receptor agonists only partially normalize hepatic gene expression in db/db mice.

Agonists of the nuclear receptors peroxisome proliferator-activated receptor (PPAR) gamma, PPARalpha, and liver X receptors (LXRs) reduce blood glucose in type 2 diabetic patients and comparable mouse models. Since the capacity of these drugs to normalize hepatic gene expression is not known, we compared groups of obese diabetic db/db mice treated with agonists for PPARgamma [rosiglitazone (Rosi); 10 mg/kg/day], PPARalpha [Wy 14643 (Wy; 4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid); 30 mg/kg/day], and LXR [T0901317 (T09; N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide); 40 mg/kg/day] and from untreated nondiabetic litter mates (db/+) by oligonucleotide microarrays and quantitative reverse transcriptase-polymerase chain reaction. The 10-day treatment period of db/db mice with Rosi, Wy, and T09 altered expression of 300, 620, and 735 genes including agonist-specific target genes, respectively. However, from the 337 genes differentially regulated in untreated db/+ versus db/db animals, only 34 (10%), 51 (15%), and 82 (24%) were regulated in the direction of the db/+ group by Rosi, Wy, and T09, respectively. Gene expression normalization by drug treatment involved glucose homeostasis, lipid homeostasis, and local glucocorticoid activation. In addition, our data pointed to hitherto unknown interference of these nuclear receptors with growth hormone receptor gene expression and endoplasmic reticulum stress. However, many diabetes-associated gene alterations remained unaffected or were even aggravated by nuclear receptor agonist treatment. These results suggest that diabetes-induced gene expression is minimally reversed by potent blood glucose-lowering nuclear receptor agonists.

Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing.

Expression of the small heat shock protein alphaB-crystallin in differentiated thyroid tumors has been described recently. In this study, we investigated the molecular mechanisms that affect the expression of alphaB-crystallin in benign goiters (n = 7) and highly malignant anaplastic thyroid carcinomas (ATCs) (n = 3). AlphaB-crystallin expression was compared with that of Hsp27-1. Immunoblot and quantitative real-time (RT) polymerase chain reaction revealed marked downregulation of alphaB-crystallin in all the tested ATCs and the ATC-derived cell line C-643 . In contrast, considerable expression of Hsp27-1 in benign and malignant thyroid tissue was demonstrated. Immunofluorescence analysis revealed no relevant topological differences between benign and malignant thyrocytes in the cytoplasmic staining of both proteins. Consistent and marked downregulation of TFCP2L1 was identified as one of the main mechanisms contributing to CRYAB gene silencing in ATCs. In addition, CRYAB gene promoter methylation seems to occur in distinct ATCs. In silico analysis revealed that the differential expression of alphaB-crystallin and Hsp27-1 results from differences between the alphaB-crystallin and Hsp27-1 promoter fragments (712 bp upstream from the transcriptional start site). Biological activity of the analyzed promoter element is confirmed by its heat shock inducibility. In conclusion, we demonstrate downregulation of alphaB-crystallin expression in highly dedifferentiated ATCs because of a tumor-specific transcription factor pattern. The differential expression of alphaB-crystallin and Hsp27-1 indicates functional differences between both proteins.