Cell Glucose Utilization Research Articles

Impact of gene modification of phosphotransferase system on expression of glutamate dehydrogenase protein of Streptococcus suis in Escherichia coli

ABSTRACTGlutamate dehydrogenase (GDH) protein of Streptococcus suis can be used for detection of S. suis infection and protection of pigs against S. suis infection. Acetate is a primary inhibitory metabolite for cell growth and formation of GDH protein. In this study, the ptsG gene, which encodes the integral membrane permease IICBGlc in the phosphotransferase system, was deleted and the effect of this deletion on the expression of GDH protein was investigated. The plasmids containing glfZ. mobilis (encoding glucose facilitator)–glkE. coli (encoding glucokinase) or galPE. coli (encoding galactose permease)–glkE. coli were transformed into ptsG mutant cells to recover the cell growth and glucose utilization of ptsG mutant. The mutants with deletion of ptsG decreased the accumulation of acetate; and higher cell density and GDH protein concentration were obtained with the ptsG mutants containing glf–glk or galP–glk. When the ptsG mutant containing glf–glk (SSGGFK) was used for expression of GDH protein, the cell density (optical density OD600 of 2.68) and the concentration of GDH protein (42.34 mg/L) were highest, with an increase by 12.61% and 14.84%, respectively, compared with the parental strain (SSG). The acetate accumulation was reduced to 2.35 g/L, i.e. a 37.33% decrease compared with the SSG strain. High concentration of GDH protein was obtained with reduction of acetate accumulation through gene modification of the phosphotransferase system. This can decrease the production cost of the subunit vaccine of GDH protein and provide theoretical foundation for high-level expression of other recombinant proteins.

Improving the production of acetyl-CoA-derived chemicals in Escherichia coli BL21(DE3) through iclR and arcA deletion

BackgroundAcetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals.ResultsIn this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn’t gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future.ConclusionsKnockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.

4-Fluoro-N'-(2-hydroxy-3-methoxybenzylidene) benzohydrazide and its Oxidovanadium(V) Complex: Syntheses, Crystal Structures and Insulin-enhancing Activity.

A hydrated hydrazone compound, 4-fluoro-N'-(2-hydroxy-3-methoxybenzylidene)benzohydrazide monohydrate (H2L · H2O), was prepared and characterized by elemental analysis, HRMS, IR, UV-Vis and 1H NMR spectroscopy. Reaction of H2L, kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one; Hka) and VO(acac)2 in methanol afforded a novel oxidovanadium(V) complex, [VO(ka)L]. The complex was characterized by elemental analysis, IR, UV-Vis and 1H NMR spectroscopy. Thermal analysis was also performed. Structures of H2L and the complex were further confirmed by single crystal structural X-ray diffraction. The vanadium complex is the first structurally characterized vanadium complex of kojic acid. Insulin-mimetic tests on C2C12 muscle cells indicate that the complex significantly stimulated cell glucose utilization with cytotoxicity at 0.11 g L-1.

Synthesis, Crystal Structures, and Insulin Enhancement of Vanadium(V) Complexes Derived From 2-Bromo-N’-(2-hydroxybenzylidene)benzohydrazide

Two new mononuclear vanadium(V) complexes with the formula [VOLX] [X = benzohydroxamate for 1, and X = 8-hydroxyquinoline for 2; L = 2-bromo-N’-(2-hydroxybenzylidene)benzohydrazide], have been prepared. The complexes have been characterized by physicochemical methods and single crystal X-ray determination. The V atoms in both complexes are coordinated by the three donor atoms of L, two donor atoms of X, and one oxo group, forming octahedral coordination. Insulin-mimetic tests on C2C12 muscle cells using biovision glucose assay indicates that the complexes significantly stimulated cell glucose utilization with cytotoxicity at 0.10 g L−1.

Improvements of metabolites tolerance in Clostridium acetobutylicum by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.

Kinetics and modelling of batch fermentation for the production of organic solvent tolerant and thermostable lipase by recombinant E. coli / Organik çözücü toleranslı ve ısıya dayanıklı rekombinan E. coli lipaz üretiminin kinetiği ve grup fermentasyonu modellemesi

Abstract Objective: Kinetics of organic solvent tolerant and thermostable lipase production by recombinant E. coli in shake flask level and 2 L stirred tank bioreactor level was studied to observe the variations of important kinetic parameters at two different levels of bioprocess. Methods: Unstructured models based on Monod equation for growth and Luedeking-Piret equation for lipase production and glucose consumption were used to predict cell growth, lipase production and glucose utilization. The shake flask fermentation experiments were carried out at different initial glucose and yeast extract concentrations using recombinant bacterial strain E. coli BL21. Lipase production was also carried out using 2L stirred tank bioreactor for comparison. Results: In all cases, the data fitted well to the proposed models. The highest growth and lipase activity were obtained at 25 g/L glucose and 25 g/L yeast extract. Cell growth (6.42 g/L) and lipase production (65.32 IU/mL) in 2 L stirred tank bioreactor was comparable to those obtained in shake flask fermentations. The calculated value of growth associated constant (9.874 IU/g/h) was much higher than that of non-growth associated constant (0.022 IU/g/h) in bioreactor as well as in shake flasks. The values of maximum specific growth rate (μm) and glucose saturation constant (KS) for shake flask fermentations, calculated from Monod equation, were 0.476 h-1 and 5.237 g/L respectively. Conclusion: From the modelling exercise, it was concluded that the lipase production is dominantly growth associated process. The kinetic parameter values for fermentations in shake flask and 2L stirred tank bioreactor were comparable, indicating that the bioprocess could be transferred into larger scale.

Synthesis, characterization, and insulin-enhancing of vanadium(V) complexes with similar Schiff base ligands

Two new mononuclear vanadium(V) complexes, [VOL1L′] (I) and [VOL2L′] (II), where L1 and L2 are the dianionic form of 2-hydroxy-N′-(4-oxopentan-2-ylidene)benzohydrazide and 4-bromo-N′-(4-oxo-pentan-2-ylidene)benzohydrazide, respectively, and L′ is 8-hydroxyquinoline, have been prepared. The complexes have been characterized by physico-chemical methods and single crystal X-ray determination (CIF files CCDC nos. 1044151 (I) and 1044152 (II)). The V atoms in both complexes are coordinated by the three donor atoms of L, two donor atoms of L′, and one oxo group, forming octahedral coordination. Insulinmimetic tests on C2C12 muscle cells using biovision glucose assay indicates that the complexes significantly stimulated cell glucose utilization with cytotoxicity at 0.10 g L–1.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose in nutrient-rich or minimal media anaerobically. Co-expression of heterologous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in BA002 resulted in a significant increase in cell mass and succinic acid production. Nevertheless, the resultant strain, BA016, still could not grow in a defined medium without tryptone and yeast extract. To solve the problem, a novel atmospheric and room temperature plasma mutation method was employed to generate mutants which can grow in the defined medium. A mutant designated as LL016 was observed to be the best strain that regained the capacity of cell growth and glucose utilization in a defined medium anaerobically. After 120h of fermentation in the defined medium, 35.0g/L of glucose was consumed to generate 25.2g/L of succinic acid. Furthermore, with the highest glucose consumption rate in the dual-phase fermentation, the yield of succinic acid in LL016 reached 0.87g/g, which was higher than that observed in other strains. From an industrial standpoint, the defined medium is much cheaper than LB medium, which shows a great potential usage for the economical production of succinic acid by LL016.

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065h−1 of dilution rate, 180g/L of feeding glucose concentration, and 16h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92g/L of produced 2KGA concentration, 8.83g/L.h of average volumetric productivity, and 0.9510g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.

Dichotomous roles for the orphan nuclear receptor NURR1 in breast cancer

BackgroundNR4A orphan nuclear receptors are involved in multiple biological processes which are important in tumorigenesis such as cell proliferation, apoptosis, differentiation, and glucose utilization. The significance of NR4A family member NURR1 (NR4A2) in breast cancer etiology has not been elucidated. The purpose of this study was to ascertain the impact of NURR1 expression on breast transformation, tumor growth, and breast cancer patient survival.MethodsWe determined the expression of NURR1 in normal breast versus breast carcinoma in tissue microarrays (immunohistochemistry), tissue lysates (immunoblot), and at the mRNA level (publically available breast microarrays). In addition NURR1 expression was compared among breast cancer patients in cohorts based on p53 expression, estrogen receptor α expression, tumor grade, and lymph node metastases. Kaplan-Meier survival plots were used to determine the correlation between NURR1 expression and relapse free survival (RFS). Using shRNA-mediated silencing, we determined the effect of NURR1 expression on tumor growth in mouse xenografts.ResultsResults from breast cancer tissue arrays demonstrate a higher NURR1 expression in the normal breast epithelium compared to breast carcinoma cells (p ≤ 0.05). Among cases of breast cancer, NURR1 expression in the primary tumors was inversely correlated with lymph node metastases (p ≤ 0.05) and p53 expression (p ≤ 0.05). Clinical stage and histological grade were not associated with variation in NURR1 expression. In gene microarrays, 4 of 5 datasets showed stronger mean expression of NURR1 in normal breast as compared to transformed breast. Additionally, NURR1 expression was strongly correlated with increase relapse free survival (HR = 0.7) in a cohort of all breast cancer patients, but showed no significant difference in survival when compared among patients whom have not been treated systemically (HR = 0.91). Paradoxically, NURR1 silenced breast xenografts showed significantly decreased growth in comparison to control, underscoring a biphasic role for NURR1 in breast cancer progression.ConclusionsNURR1 function presents a dichotomy in breast cancer etiology, in which NURR1 expression is associated with normal breast epithelial differentiation and efficacy of systemic cancer therapy, but silencing of which attenuates tumor growth. This provides a strong rationale for the potential implementation of NURR1 as a pharmacologic target and biomarker for therapeutic efficacy in breast cancer.

Regulation of NAD(H) pool and NADH/NAD+ ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD+ regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD+ ratio from 0.64 to 0.13, in particular, the concentration of NAD+ increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD+ for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD+ ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD+ ratio was very important for succinic acid production.

Application of Central Composite Design and Artificial Neural Network for the Optimization of Fermentation Conditions for Lipase Production by Rhizopus arrhizus MTCC 2233

The response surface optimization strategy was used to enhance the lipase production by Rhizopus arrhizus MTCC 2233 in submerged fermentation. Various vegetable oils were experimented as an inducer using the optimized medium to study the influence on lipase production, and corn oil was found to be the best inducer for lipase production by Rhizopus arrhizus. The optimization of fermentation conditions, temperature, initial pH and agitation speed was carried out using corn oil as the inducer. Statistical analysis of the experimental data showed that the temperature, agitation speed, quadratic effects of temperature, initial pH and agitation speed and interactive effects of temperature and agitation speed are significant parameters that affect lipase production. The optimum fermentation conditions were achieved at 32°C; pH 6.0 and agitation speed of 107 rpm with the maximum lipase activity of 4.32 U/mL. Artificial neural network model was used to predict the lipase activity and cell mass production under various fermentation conditions. Unstructured kinetic models, Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were used to describe the cell biomass, lipase production and glucose utilization kinetics respectively.

The Effect of Aspirin on Red Cell Electrolyte, ATP Levels and Glucose Utilization in Rats

Wistar rats were fed chow supplemented with 1% (w/w) aspirin for one month. At 3, 7, 14, 21 and 30 days of treatment, heparinized blood was collected. ATP, Na+, K+ levels, glucose utilization of erythrocytes and inorganic phosphate, Na+, K+, salicylate levels of plasma were determined. Erythrocyte ATP and plasma phosphate levels were decreased following aspirin treatment. ATP decrement caused erythrocyte K+ level to diminish significantly. Plasma Na+ and K+ levels were not effected by aspirin treatment. In addition, the decrease in ATP levels induced erythrocyte glucose utilization. It was concluded that the therapeutic dose of aspirin in man, gave rise to a decrease in the energy production and an impairment of energy utilizing events in erythrocytes in rats (the therapeutic serum aspirin concentration in man is 25-40 mg dL−1).

Synthesis, characterization, and insulin-enhancing studies of unsymmetrical tetradentate Schiff-base complexes of oxovanadium(IV)

A series of unsymmetrical tetradentate Schiff bases were synthesized by interaction of 2-hydroxy-1-naphthaldehyde, phenylenediamine and salicylaldehyde, or substituted salicylaldehyde in an ethanolic medium. The oxovanadium(IV) complexes and the ligands were synthesized and characterized by elemental analyses, 1H NMR, infrared, electron paramagnetic resonance, electronic spectra, cyclic voltammetry, and room temperature magnetic susceptibility measurements. The elemental analyses for both the ligands and the metal complexes confirmed purity of the compounds as formulated. Electron paramagnetic resonance spectra of the complexes were measured as powder and in toluene/dichloromethane (9 : 1, v/v) solution at room and liquid N2 temperatures. The g values, g o = 1.971, g ⊥ = 1.978, and g = 1.950, are the same for all the complexes examined. The vanadium nuclear hyperfine splitting, A o = 101–99, A ⊥ = 65–64, A ∥ = 179–177, vary slightly with substituents on the salicylaldehyde. Infrared spectra reveal strong V=O stretching bands in the range 970–988 cm−1, typical of monomeric five-coordinate complexes. The room temperature magnetic moments of 1.6–1.8 BM for the complexes confirmed that the complexes are V(IV) complexes, with d1 configuration. Only one quasi-reversible wave is observed for each compound and they all showed redox couples with peak-to-peak separation values (ΔE p) ranging from 78 to 83 mV, indicating a single step one electron transfer process. Insulin-mimetic tests on C2C12 muscle cells using Biovision glucose assay showed that all the complexes significantly stimulated cell glucose utilization with negligible cytotoxicity at 0.05 µg µL−1.

Neuroendocrine inhibition of glucose production and resistance to cancer in dwarf mice

Pit1 null (Snell dwarf) and Proph1 null (Ames dwarf) mutant mice lack GH, PRL and TSH. Snell and Ames dwarf mice also exhibit reduced IGF-I, resistance to cancer and a longer lifespan than control mice. Endogenous glucose production during fasting is reduced in Snell dwarf mice compared to fasting control mice. In view of cancer cell dependence on glucose for energy, low endogenous glucose production may provide Snell dwarf mice with resistance to cancer. We investigated whether endogenous glucose production is lower in Snell dwarf mice during feeding. Inhibition of endogenous glucose production by glucose injection was enhanced in 12 to 14 month-old female Snell dwarf mice. Thus, we hypothesize that lower endogenous glucose production during feeding and fasting reduces cancer cell glucose utilization providing Snell dwarf mice with resistance to cancer. The elevation of circulating adiponectin, a hormone produced by adipose tissue, may contribute to the suppression of endogenous glucose production in 12 to 14 month-old Snell dwarf mice. We compared the incidence of cancer at time of death between old Snell dwarf and control mice. Only 18% of old Snell dwarf mice had malignant lesions at the time of death compared to 82% of control mice. The median ages at death for old Snell dwarf and control mice were 33 and 26 months, respectively. By contrast, previous studies showed a high incidence of cancer in old Ames dwarf mice at the time of death. Hence, resistance to cancer in old Snell dwarf mice may be mediated by neuroendocrine factors that reduce glucose utilization besides elevated adiponectin, reduced IGF-I and a lack of GH, PRL and TSH, seen in both Snell and Ames dwarf mice. Proteomics analysis of pituitary secretions from Snell dwarf mice confirmed the absence of GH and PRL, the secretion of ACTH and elevated secretion of Chromogranin B and Secretogranin II. Radioimmune assays confirmed that circulating Chromogranin B and Secretogranin II were elevated in 12 to 14 month-old Snell dwarf mice. In summary, our results in Snell dwarf mice suggest that the pituitary gland and adipose tissue are part of a neuroendocrine loop that lowers the risk of cancer during aging by reducing the availability of glucose.

Renal cell carcinoma

The recent contributions to renal cell carcinoma in the fields of molecular biology and the expanded use of molecularly targeted agents will be reviewed. This study is intended to update prognostic and therapeutic decision-making data and provide perspective on advances in understanding the molecular biology of this disease. Updates to the currently used prognostic algorithms for renal cell carcinoma are needed, and recently verified prognostic nomograms will be discussed. This comes in the wake of numerous advances in the use of molecularly targeted drugs, which will be reviewed. Finally, advancements in understanding the biology of renal cell carcinoma include the discovery of von Hippel-Lindau associated mechanisms involved in renal cyst formation and renewed appreciation for the influence of this pathway on the tumor cell glucose utilization profile. Renal cell carcinoma continues to evolve swiftly with the approval of new agents and the maturation of clinical trials to provide relevant structure to treatment decisions. This study will give an overview of the latest concepts in the epidemiology and biology of renal cell carcinoma and provide current surgical and systemic updates for managing renal cell carcinoma.

An optimized in vitro assay for screening compounds that stimulate liver cell glucose utilization with low cytotoxicity

Introduction Screening compounds that stimulate liver cell glucose utilization with low cytotoxicity plays an important role in anti-diabetic drug discovery. Existing in vitro assay for the quantitative measurement of cell glucose utilization rate and cytotoxicity of compounds needs to be modified in order to improve the simplicity and reproducibility. An optimized assay was described to addresses these problems. Methods Compounds were directly mixed with cell suspension. After 20 h incubation, glucose decrement detection in culture medium was performed, followed by the assessment of cell viability using Cell Counting Kit-8 (CCK-8). Results A series of experiments were conducted to define optimal conditions such as cell inoculation density, incubation time and CCK-8 concentration in cell culture medium. Glucose utilization unit (GUU) was defined for the evaluation of both efficacy and cytotoxicity of a compound. It was found that Rosiglitazone significantly stimulated cell glucose utilization with negligible cytotoxicity at the concentration of 10 − 5 M. Conclusion The simplicity and efficiency of the optimized method has been proven in this study. Our findings show that this assay has potential application in anti-diabetic drug discovery such as new peroxisome proliferator-activated receptor γ (PPARγ) agonists.

FoxO1 Stimulates Fatty Acid Uptake and Oxidation in Muscle Cells through CD36-dependent and -independent Mechanisms

Emerging evidence documents a key function for the forkhead transcription factor FoxO1 in cellular metabolism. Here, we investigate the role of FoxO1 in the regulation of fatty acid (FA) metabolism in muscle cells. C2C12 cells expressing an inducible construct with either wild type FoxO1 or a mutant form (FoxO1/TSS) refractory to the protein kinase B inhibitory effects were generated. FoxO1 activation after myotube formation altered the expression of several genes of FA metabolism. Acyl-CoA oxidase and peroxisome proliferator-activated receptor delta mRNA levels increased 2.2-fold and 1.4-fold, respectively, whereas mRNA for acetyl-CoA carboxylase decreased by 50%. Membrane uptake of oleate increased 3-fold, and oleate oxidation increased 2-fold. Cellular triglyceride content was also increased. The enhanced FA utilization induced by FoxO1 was mediated by a severalfold increase in plasma membrane level of the fatty acid translocase FAT/CD36 and eliminated by cell treatment with the CD36 inhibitor sulfo-N-succinimidyl-oleate. We conclude that FoxO1 activation induces coordinate increases in FA uptake and oxidation and that these effects are mediated, at least in part, by membrane enrichment in CD36. The data suggest that FoxO1 contributes to preparing the muscle cell for the increased reliance on FA metabolism that is characteristic of fasting. Dysregulation of FoxO1 in muscle could contribute to intramuscular lipid accumulation and insulin resistance by maintaining activation of FA uptake.

Enhanced rat β-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway

Islet beta-cell proliferation is a very important component of beta-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of beta-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of beta-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only approximately 1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.