The presence of pyruvate carboxylase in the human brain and its role in the survival of cultured human astrocytes.

Pyruvate carboxylase (PC) is a mitochondrial, biotin-containing enzyme catalyzing the ATP-dependent synthesis of oxaloacetate from pyruvate and bicarbonate, with a critical anaplerotic role in sustaining the brain metabolism. Based on the studies performed on animal models, PC expression was assigned to be glia-specific. To study PC distribution among human neural cells, we probed the cultured human astrocytes and brain sections with antibodies against PC. Additionally, we tested the importance of PC for the viability of cultured human astrocytes by applying the PC inhibitor 3-chloropropane-1,2-diol (CPD). Our results establish the expression of PC in mitochondria of human astrocytes in culture and brain tissue and also into a subpopulation of the neurons in situ. CPD negatively affected the viability of astrocytes in culture, which could be partially reversed by supplementing media with malate, 2-oxoglutarate, citrate, or pyruvate. The provided data estimates PC expression in human astrocytes and neurons in human brain parenchyma. Furthermore, the enzymatic activity of PC is vital for sustaining the viability of cultured astrocytes.

Erianin suppresses proliferation and migration of cancer cells in a pyruvate carboxylase-dependent manner.

Erianin is a natural small molecule dibenzyl compound extracted from Dendrobium officinale or Dendrobium chrysotoxum. Studies show erianin has many pharmacological functions such as antioxidant, antibacterial, antiviral, improving diabetic nephropathy, relaxing bronchial smooth muscle and anti-tumor. However, the erianin-mediated molecular mechanism is elusive, and the target protein of erianin is not clear yet. Here, we screened and identified that the target protein of erianin in human hepatoma HepG2 cells is human pyruvate carboxylase, and explored the anti-tumor signal pathway regulated by erianin in several cell lines. Firstly, the interaction between human pyruvate carboxylase and erianin was studied by bioinformatics and biochemical methods. Secondly, in vitro, erianin can specifically inhibit the activity of human pyruvate carboxylase, and the purified human pyruvate carboxylase can specifically bind to the activity probe of erianin. Thirdly, human pyruvate carboxylase is highly expressed in a variety of malignant tumors, and the inhibitory effect of erianin on tumor cells is positively correlated with the expression of human pyruvate carboxylase, and erianin can selectively inhibit the activity of pyruvate carboxylase. Finally, erianin can regulate the pyruvate carboxylase-mediated Wnt/ ß- Catenin pathway. All of which provide important data for the further study of the anticancer mechanism of erianin, and lay a solid foundation for the further development and utilization of erianin.

The Flavonoid Kaempferol Ameliorates Streptozotocin-Induced Diabetes by Suppressing Hepatic Glucose Production.

In diabetes mellitus, the excessive rate of glucose production from the liver is considered a primary contributor for the development of hyperglycemia, in particular, fasting hyperglycemia. In this study, we investigated whether kaempferol, a flavonol present in several medicinal herbs and foods, can be used to ameliorate diabetes in an animal model of insulin deficiency and further explored the mechanism underlying the anti-diabetic effect of this flavonol. We demonstrate that oral administration of kaempferol (50 mg/kg/day) to streptozotocin-induced diabetic mice significantly improved hyperglycemia and reduced the incidence of overt diabetes from 100% to 77.8%. This outcome was accompanied by a reduction in hepatic glucose production and an increase in glucose oxidation in the muscle of the diabetic mice, whereas body weight, calorie intake, body composition, and plasma insulin and glucagon levels were not altered. Consistently, treatment with kaempferol restored hexokinase activity in the liver and skeletal muscle of diabetic mice while suppressed hepatic pyruvate carboxylase activity and gluconeogenesis. These results suggest that kaempferol may exert antidiabetic action via promoting glucose metabolism in skeletal muscle and inhibiting gluconeogenesis in the liver.

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions.

Inorganic phosphate (Pi ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi , adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13 C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.

Cloning and characterization of pyruvate carboxylase gene responsible for calcium malate overproduction in Penicillium viticola 152 and its expression analysis.

In this study, a pyruvate carboxylase gene (PYC) from a marine fungus Penicillium viticola 152 isolated from marine algae was cloned and characterized by using Genome Walking method. An open reading frame (ORF) of The PYC gene (accession number: KM593097) had 3582bp encoding 1193 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 131.2757kDa. A putative promoter (intronless) of the gene was located at -666bp and contained a TATA box, several CAAT boxes, the 5'-SYGGRG-3' and a 5'-HGATAR-3' sequences. A consensus polyadenylation site (AATAAA) was also observed at +10bp downstream of the ORF. The protein deduced from the PYC gene had no signal peptide, was a homotetramer (4), and had the four functional domains. Furthermore, PYC protein also had three potential N-linked glycosylation sites, among them, -N-S-T-I- at 36 amino acid, -N-G-T-V- at 237 amino acid, and -N-G-S-S- at 517 amino acid were the most possible N-glycosylation sites. After expression of the PYC gene of P. viticola 152 in medium supplemented with CSL and biotin, it was found that the specific pyruvate carboxylase activity in MA production medium supplemented with CSL was much higher (0.5U/mg) than in MA medium supplemented with biotin (0.3U/mg), suggesting that optimal concentration of CSL is required for increased expression of the PYC gene, which is responsible for high level production of malic acid in P. viticola 152 strain.

Feeding glycerol-enriched yeast culture improves lactation performance, energy status, and hepatic gluconeogenic enzyme expression of dairy cows during the transition period.

This study aimed to evaluate the effects of feeding glycerol-enriched yeast culture (GY) on feed intake, lactation performance, blood metabolites, and expression of some key hepatic gluconeogenic enzymes in dairy cows during the transition period. Forty-four multiparous transition Holstein cows were blocked by parity, previous 305-d mature equivalent milk yield, and expected calving date and randomly allocated to 4 dietary treatments: Control (no additive), 2 L/d of GY (75.8 g/L glycerol and 15.3 g/L yeast), 150 g/d of glycerol (G; 0.998 g/g glycerol), and 1 L/d of yeast culture (Y; 31.1 g/L yeast). All additives were top-dressed and hand mixed into the upper one-third of the total mixed ration in the morning from -14 to +28 d relative to calving. Results indicated that the DMI, NE intake, change of BCS, and milk yields were not affected by the treatments ( > 0.05). Supplementation of GY or Y increased milk fat percentages, milk protein percentages, and milk protein yields relative to the Control or G group ( < 0.05). Cows fed GY or G had higher glucose levels and lower ß-hydroxybutyric acid (BHBA) and NEFA levels in plasma than cows fed the Control ( < 0.05) and had lower NEFA levels than cows fed Y ( < 0.05). On 14 d postpartum, cows fed GY or G had higher enzyme activities, mRNA, and protein expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; < 0.05); higher enzyme activities ( < 0.05) and a tendency toward higher mRNA expression ( < 0.10) of glycerol kinase (GK); and a tendency toward higher enzyme activities of pyruvate carboxylase (PC) in the liver ( < 0.10) when compared with cows fed Control or Y. The enzyme activities, mRNA, and protein expression of PEPCK-C, PC, and GK did not differ between cows fed GY and G ( > 0.10). In conclusion, dietary GY or Y supplementation increased the milk fat and protein content of the cows in early lactation and GY or G supplementation improved the energy status as indicated by greater plasma glucose and lower plasma BHBA and NEFA concentrations and upregulated the hepatic gluconeogenic enzymes of dairy cows during the transition period. Feeding cows with a GY mixture in the peripartum period combined the effects of yeast on lactation performance and the effects of glycerol on energy status in dairy cows.

Short communication: Regulation of hepatic gluconeogenic enzymes by dietary glycerol in transition dairy cows.

Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from -28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28, -14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production.

Biotin deprivation impairs mitochondrial structure and function and has implications for inherited metabolic disorders.

Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect,whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases.Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.

Effect of sorghum grain supplementation on glucose metabolism in cattle and sheep fed temperate pasture.

The aim of this work was to evaluate the effect of sorghum grain supplementation on plasma glucose, insulin and glucagon concentrations, and hepatic mRNA concentrations of insulin receptor (INSR), pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PCK1) mRNA and their association with nutrient intake, digestion and rumen volatile fatty acids (VFA) in cattle and sheep fed a fresh temperate pasture. Twelve Hereford × Aberdeen Angus heifers and 12 Corriedale × Milchschaf wethers in positive energy balance were assigned within each species to one of two treatments (n = 6 per treatment within specie): non-supplemented or supplemented with sorghum grain at 15 g/kg of their body weight (BW). Supplemented cattle had greater plasma glucose concentrations, decreased plasma glucagon concentrations and tended to have greater plasma insulin and insulin-to-glucagon ratio than non-supplemented ones. Hepatic expression of INSR and PC mRNA did not differ between treatments but PCK1 mRNA was less in supplemented than non-supplemented cattle. Supplemented sheep tended to have greater plasma glucagon concentrations than non-supplemented ones. Plasma glucose, insulin, insulin-to-glucagon ratio, and hepatic expression of INSR and PC mRNA did not differ between treatments, but PCK1 mRNA was less in supplemented than non-supplemented sheep. The inclusion of sorghum grain in the diet decreased PCK1 mRNA but did not affect PC mRNA in both species; these effects were associated with changes in glucose and endocrine profiles in cattle but not in sheep. Results would suggest that sorghum grain supplementation of animals in positive energy balance (cattle and sheep) fed a fresh temperate pasture would modify hepatic metabolism to prioritize the use of propionate as a gluconeogenic precursor.

Cod liver oil ameliorates sodium nitrite-induced insulin resistance and degradation of rat hepatic glycogen through inhibition of cAMP/PKA pathway.

AIMS: Sodium nitrite is used to inhibit the growth of microorganisms and is responsible for the desirable red color of meat; however, it can be toxic in high quantities for humans and other animals. Moreover, glycogen, a branched polysaccharide, efficiently stores and releases glucose monosaccharides to be accessible for metabolic and synthetic requirements of the cell. Therefore, we examined the impact of dietary sodium nitrite and cod liver oil on liver glycogen. MAIN METHODS: Thirty-two Sprague-Dawley rats were treated daily with sodium nitrite (80 mg/kg) in the presence/absence of cod liver oil (5 ml/kg). Liver sections were stained with Periodic acid-Schiff. Hepatic homogenates were used for measurements of glycogen, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), glycogen synthase, glycogen synthase kinase, pyruvate carboxylase, fructose 1,6-diphosphatase, glucose 6-phosphatase, phosphodiesterase and glycogen phosphorylase. Glucose, pyruvate tolerances and HOMA insulin resistance were also determined. KEY FINDINGS: Sodium nitrite significantly increased plasma glucose and insulin resistance. Moreover, sodium nitrite significantly reduced hepatic glycogen content as well as activities of glycogen synthase, glycogen synthase kinase-3, and phosphodiesterase. Sodium nitrite elevated hepatic cAMP, PKA, pyruvate carboxylase, fructose 1,6-diphosphatase, glucose 6-phosphatase and phosphorylase. Cod liver oil significantly blocked all of these except pyruvate carboxylase, fructose 1,6-diphosphatase and glucose 6-phosphatase. SIGNIFICANCE: Sodium nitrite inhibited liver glycogenesis and enhanced liver glycogenolysis and gluconeogenesis, which is accompanied by hyperglycemia and insulin resistance through the activation of cAMP/PKA and the inhibition of phosphodiesterase. Cod liver oil blocked the sodium nitrite effects on glycogenesis and glycogenolysis without affecting gluconeogenesis.

Moringa oleifera leaf extract ameliorates alloxan-induced diabetes in rats by regeneration of ß cells and reduction of pyruvate carboxylase expression.

Moringa oleifera Lam. contains many active ingredients with nutritional and medicinal values. It is commonly used in folk medicine as an antidiabetic agent. The present study was designed to investigate how an aqueous extract from the leaves of M. oleifera reveals hypoglycemia in diabetic rats. M. oleifera leaf extract counteracted the alloxan-induced diabetic effects in rats as it normalized the elevated serum levels of glucose, triglycerides, cholesterol, and malondialdehyde, and normalized mRNA expression of the gluconeogenic enzyme pyruvate carboxylase in hepatic tissues. It also increased live body weight gain and normalized the reduced mRNA expression of fatty acid synthase in the liver of diabetic rats. Moreover, it restored the normal histological structure of the liver and pancreas damaged by alloxan in diabetic rats. This study revealed that the aqueous extract of M. oleifera leaves possesses potent hypoglycemic effects through the normalization of elevated hepatic pyruvate carboxylase enzyme and regeneration of damaged hepatocytes and pancreatic ß cells via its antioxidant properties.

Effects of biotin supplementation in the diet on insulin secretion, islet gene expression, glucose homeostasis and beta-cell proportion.

Besides its role as a carboxylase cofactor, biotin has a wide repertoire of effects on gene expression, development and metabolism. Pharmacological concentrations of biotin enhance insulin secretion and the expression of genes and signaling pathways that favor islet function in vitro. However, the in vivo effects of biotin supplementation on pancreatic islet function are largely unknown. In the present study, we investigated whether in vivo biotin supplementation in the diet has positive effects in rodent pancreatic islets. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet over 8 weeks postweaning and tested for glucose homeostasis, insulin secretion, islet gene expression and pancreatic morphometry. Insulin secretion increased from the islets of biotin-supplemented mice, together with the messenger RNA (mRNA) expression of several transcription factors regulating insulin expression and secretion, including forkhead box A2, pancreatic and duodenal homeobox 1 and hepatocyte nuclear factor 4α. The mRNA abundance of glucokinase, Cacna1d, acetyl-CoA carboxylase, and insulin also increased. Consistent with these effects, glucose tolerance improved, and glucose-stimulated serum insulin levels increased in biotin-supplemented mice, without changes in fasting glucose levels or insulin tolerance. Biotin supplementation augmented the proportion of beta cells by enlarging islet size and, unexpectedly, also increased the percentage of islets with alpha cells at the islet core. mRNA expression of neural cell adhesion molecule 1, an adhesion protein participating in the maintenance of islet architecture, decreased in biotin-supplemented islets. These findings provide, for the first time, insight into how biotin supplementation exerts its effects on function and proportion of beta cells, suggesting a role for biotin in the prevention and treatment of diabetes.

Rosmarinus officinalis polyphenols produce anti-depressant like effect through monoaminergic and cholinergic functions modulation.

Rosmarinus officinalis (R. officinalis), a culinary aromatic and medicinal plant, is very rich in polyphenols and flavonoids with high antioxidant properties. This plant was reported to exert multiple benefits for neuronal system and alleviate mood disorder. In our previous study, we demonstrated that R. officinalis and its active compounds, luteolin (Lut), carnosic acid (CA), and rosmarinic acid (RA), exhibited neurotrophic effects and improved cholinergic functions in PC12 cells in correlation with mitogen-activated protein kinase (MAPK), ERK1/2 signaling pathway. The current study was conducted to evaluate and understand the anti-depressant effect of R. officinalis using tail suspension test (TST) in ICR mice and PC12 cells as in vitro neuronal model. Proteomics analysis of PC12 cells treated with R. officinalis polyphenols (ROP) Lut, CA, and RA revealed a significant upregulation of tyrosine hydroxylase (TH) and pyruvate carboxylase (PC) two major genes involved in dopaminergic, serotonergic and GABAergic pathway regulations. Moreover, ROP were demonstrated to protect neuronal cells against corticosterone-induced toxicity. These results were concordant with decreasing immobility time in TST and regulation of several neurotransmitters (dopamine, norepinephrine, serotonin and acetylcholine) and gene expression in mice brain like TH, PC and MAPK phosphatase (MKP-1). To the best of our knowledge this is the first evidence to contribute to the understanding of molecular mechanism behind the anti-depressant effect of R. officinalis and its major active compounds.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Characterization of bovine pyruvate carboxylase promoter 1 responsiveness to serum from control and feed-restricted cows.

Pyruvate carboxylase (PC; EC 6.4.1.1) is critical in gluconeogenesis from lactate and maintenance of tricarboxylic acid cycle intermediates. Whereas increases in PC mRNA have been observed during feed restriction, the mechanism of regulation is unknown; however, coinciding increases in circulating NEFA concentrations suggests that fatty acids may contribute to regulation of gene expression during feed restriction. The objective of this study was to examine the direct effect of exposure to serum from full-fed control cows with serum from cows that were restricted to 50% of ad libitum intake for 5 d on PC expression in vitro. Rat hepatoma (H4IIE) cells were transiently transfected with bovine promoter-luciferase constructs containing bovine PC promoter 1 and treated with serum from control cows, serum from feed-restricted cows, or modified serum. Modified serum pools were generated by supplemented serum from control cows with C14:0, C16:0, C18:0, C18:1n-9 cis, C18:2n-6 cis, and C18:3n-3 cis to match the total NEFA in serum from feed-restricted cows (1.3 mM) in the relative proportion found in serum from control or feed-restricted cows. Exposure of cells to serum from feed-restricted cows increased (P < 0.05) PC promoter 1 activity 2.2-fold compared with cells exposed to control cow serum. Exposure to serum from control cows with fatty acids added to a NEFA concentration of 1.3 mM to reflect the fatty acid profile of control and feed-restricted cows increased (P < 0.05) promoter 1 activity 2.1- and 2.5-fold, respectively, compared with cells incubated with control cow serum. There was no difference (P ≥ 0.05) in promoter 1 activity in cells treated with modified serum compared with serum from feed-restricted cows. These data indicate that promoter 1 is activated by fatty acids found in serum of feed-restricted cows. These data suggest a role of NEFA to regulate expression of bovine PC mRNA through specific activation of PC promoter 1.

Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an acetylated variant, N-acetyl-diaminopentane, which reached levels of more than 25% of that of the desired product.

Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.

Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme's properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.

Biotin deficiency affects both synthesis and degradation of pyruvate carboxylase in rat primary hepatocyte cultures.

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that plays a crucial role in gluconeogenesis, lipogenesis, Krebs cycle anaplerosis and amino acid catabolism. Biotin deficiency reduces its mass besides its activity. Enzyme mass is the result of its cellular turnover, i.e., its rates of synthesis and degradation. We have now investigated, by a pulse and chase approach in cultured primary hepatocytes, the effects of biotin deficiency on these rates. Wistar rats were fed a biotin-deficient diet and the controls were fed the same diet supplemented with biotin; their biotin status was monitored measuring lymphocytes propionyl-CoA carboxylase activity and urinary 3-hydroxyisovaleric acid. After 6-7 weeks primary hepatocytes were cultured in biotin-deficient or complete DMEM. PC activity was determined by measuring the incorporation of (14)C-bicarbonate into acid-non-volatile products, and its mass by streptavidin Western blots. Its synthesis rate was estimated from [(35)S] methionine incorporation into anti-PC antibody immunoprecipitate. Its degradation rate was calculated from the loss of radioactivity from previously labeled hepatocytes, in a medium containing an excess of non-radioactive methionine. PC synthesis rate in biotin-deficient hepatocytes was approximately 4.5-fold lower than in the controls, and its degradation rate was 5.1-fold higher. Therefore, the decrement of PC mass during biotin deficiency results both from a decrease in its synthesis and an increase in its degradation rates. To our knowledge, this is the first instance where a mammalian enzyme cofactor is necessary to sustain both processes.

Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

Differential effects of selenium compounds on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies.

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.