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.

A unique insight for Xiaoyao San exerts antidepressant effects by modulating hippocampal glucose catabolism using stable isotope-resolved metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) theory, depression is an emotional disease, which is thought to be related to stagnation of liver qi and dysfunction of the spleen in transport. Xiaoyao San (XYS) is considered to have the effects of soothing liver-qi stagnation and invigorating the spleen. The spleen has the function to transport and transform nutrients. The liver has also termed the center of energy metabolism in the body. Therefore, exploring the antidepressant effects of XYS from the perspective of energy metabolism may reveal new findings. AIM OF THE STUDY: Glucose catabolism is an important part of energy metabolism. In recent years, several researchers have found that XYS can exert antidepressant effects by modulating abnormalities in glucose catabolism-related metabolites. The previous research of our research group found that the hippocampus glucose catabolism was disordered in depression. However, the antidepressant potential of XYS through modulating the disorders of hippocampal glucose catabolism and the specific metabolic pathways and targets of XYS action were still unknown. The aim of this study was to address the above scientific questions. MATERIALS AND METHODS: In this research, the CUMS (chronic unpredictable mild stress) model was used as the animal model of depression. The antidepressant effect of XYS was evaluated by behavioral indicators. The specific pathways and targets of XYS modulating the disorders of glucose catabolism in the hippocampus of CUMS rats were obtained by stable isotope-resolved metabolomics. Further, the isotope tracing results were also verified by molecular biology and electron transmission electron microscopy. RESULTS: The results demonstrated that XYS pretreatment could significantly improve the depressive symptoms induced by CUMS. More importantly, it was found that XYS could modulate the disorders of glucose catabolism in the hippocampus of CUMS rats. Stable isotope-resolved metabolomics and enzyme activity tests showed that Lactate dehydrogenase (LDH), Pyruvate carboxylase (PC), and Pyruvate dehydrogenase (PDH) were targets of XYS for modulating the disorders of glucose catabolism in the hippocampus of CUMS rats. The Succinate dehydrogenase (SDH) and mitochondrial respiratory chain complex V (MRCC-Ⅴ) were targets of XYS to improve abnormal mitochondrial oxidative phosphorylation in the hippocampus of CUMS rats. XYS was also found to have the ability to improve the structural damage of mitochondria and nuclei in the hippocampal caused by CUMS. CONCLUSIONS: This study was to explore the antidepressant effect of XYS from the perspective of glucose catabolism based on a strategy combining stable isotope tracing, molecular biology techniques, and transmission electron microscopy. We not only obtained the specific pathways and targets of XYS to improve the disorders of glucose catabolism in the hippocampus of CUMS rats, but also revealed the specific targets of the pathways of XYS compared with VLF.

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.

Effect of Caralluma tuberculata on regulation of carbohydrate metabolizing genes in alloxan-induced rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma tuberculata (C. tuberculata) has traditionally been used in Pakistan and other parts of the world as a folk treatment for diabetes mellitus. A few studies indicated its antihyperglycemic effect, however, the mystery remained unfolded as how did it modify the pathophysiological condition. AIM OF STUDY: Hence, this study aimed to explore underlying mechanism(s) for its hypoglycemic activity at biochemical and molecular levels. MATERIALS AND METHODS: Methanol extract (ME) of C. tuberculata as well as its hexane (HF) and aqueous (AF) fractions were explored for their effect on total glycogen in liver and skeletal muscle of alloxan-induced rats by spectroscopy. Moreover, the expression of genes related to hepatic carbohydrate metabolizing enzymes was quantified. At molecular level, mRNA expression of glucose transporter 2 (GLUT-2), glycogen synthase (GS), glucokinase (GK), hexokinase 1 (HK-1), pyruvate kinase (PK), glucose 6 phosphate dehydrogenase (G-6-PDH), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G-6-Pase) was determined by using quantitative real time polymerase chain reaction (qRT-PCR) after administration of ME (350 mg), HF(3 mg), AF (10 mg) and metformin (500 mg). The doses were administered twice daily according to per kg of body weight. RESULTS: A significant reduction in hepatic and skeletal muscle glycogen content was exhibited. The data of qRT-PCR revealed that gene's expression of GLUT-2 was significantly decreased after treatment with ME and HF, whilst it was unaltered by AF, however, a significant decrease was observed in genes corresponding to GS, GK and HK-1 after treatment with ME. Similarly, there was a significant decrease in expression of genes corresponding to GS, GK and HK-1 following treatment with HF. Surprisingly, post-treatment with AF didn't modify the gene's expression of GS and GK, whilst it caused a profound decrease in expression of HK-1 gene. Contrarily, the expression of gene related to PK was significantly up-regulated post-administration with ME, HF and AF. The expression levels of G-6-PDH, however, remained unaltered after treatment with the experimental extract and fractions of the plant. In addition, HF and AF did not cause any modification in PEPCK, whereas ME caused a significant down-regulation of the gene. Treatment with all the extract and fractions of the plant caused a substantial decrease in the gene's expression of PC, while there was a significant increase in the expression of gene related to G-6-Pase. CONCLUSION: The three experimental extract and fractions caused a substantial decrease in glycogen content in liver and skeletal muscle tissues. The analysis by qRT-PCR showed that glucose transport via GLUT-2 was profoundly declined by ME and HF. The expression of genes related to various metabolic pathways involved in metabolism of carbohydrate in hepatocytes revealed explicitly that the ME, HF and AF decreased the phenomena of glycogenesis and gluconeogenesis. Contrarily, all the extract and fractions of the plant activated glycogenolysis and glycolysis but did not modify the pentose phosphate shunt pathway.

The Chinese patent medicine, Jin-tang-ning, ameliorates hyperglycemia through improving ß cell function in pre-diabetic KKAy mice.

Jin-tang-ning (JTN), a Chinese patent medicine, mainly comprised of Bombyx moriL., has been proved to show α-glucosidase inhibitory efficacy and clinically effective for the treatment of type 2 diabetes (T2DM). Recently, we have reported that JTN could ameliorate postprandial hyperglycemia and improved ß cell function in monosodium glutamate (MSG)-induced obese mice, suggesting that JTN might play a potential role in preventing the conversion of impaired glucose tolerance (IGT) to T2DM. In this study, we evaluated the effect of JTN on the progression of T2DM in the pre-diabetic KKAy mice. During the 10 weeks of treatment, blood biochemical analysis and oral glucose tolerance tests were performed to evaluate glucose and lipid profiles. The ß cell function was quantified using hyperglycemic clamp at the end of the study. JTN-treated groups exhibited slowly raised fasting and postprandial blood glucose levels, and also ameliorated lipid profile. JTN improved glucose intolerance after 8 weeks of treatment. Meanwhile, JTN restored glucose-stimulated first-phase of insulin secretion and induced higher maximum insulin levels in the hyperglycemic clamp. Thus, to investigate the underlying mechanisms of JTN in protecting ß cell function, the morphologic changes of the pancreatic islets were observed by optical microscope and immunofluorescence of hormones (insulin and glucagon). Pancreatic protein expression levels of key factors involving in insulin secretion-related pathway and ER stress were also detected by Western blot. Pre-diabetic KKAy mice exhibited a compensatory augment in ß cell mass and abnormal α cell distribution. Long-term treatment of JTN recovered islet morphology accompanied by reducing α cell area in KKAy mice. JTN upregulated expression levels of glucokinase (GCK), pyruvate carboxylase (PCB) and pancreas duodenum homeobox-1 (PDX-1), while down-regulating C/EBP homologous protein (Chop) expression in pancreas of the hyperglycemic clamp, which indicated the improvement of mitochondrial metabolism and relief of endoplasmic reticulum (ER) stress of ß cells after JTN treatment. These results will provide a new insight into exploring a novel strategy of JTN for protecting ß cell function and preventing the onset of pre-diabetes to T2DM.

Postpartum supplementation with fermented ammoniated condensed whey altered nutrient partitioning to support hepatic metabolism.

Our previously published paper demonstrated that fermented ammoniated condensed whey (FACW) supplementation improved feed efficiency and metabolic profile in postpartum dairy cows. The objective of this study was to further explore the effects of FACW supplementation on liver triglyceride content, hepatic gene expression and protein abundance, and plasma biomarkers related to liver function, inflammation, and damage. Individually fed multiparous Holstein cows were blocked by calving date and randomly assigned to postpartum (1 to 45 d in milk, DIM) isonitrogenous treatments: control diet (n = 20) or diet supplemented with FACW (2.9% dry matter of diet as GlucoBoost; Fermented Nutrition, Luxemburg, WI, replacing soybean meal; n = 19). Liver biopsies were performed at 14 and 28 DIM for analysis of mRNA expression, protein abundance, and liver triglyceride content. There was marginal evidence for a reduction in liver triglyceride content at 14 DIM in FACW-supplemented cows compared with the control group. Cows supplemented with FACW had greater mRNA expression of glucose-6-phosphatase at 14 DIM relative to control. Supplementation with FACW increased mRNA expression of pyruvate carboxylase (PC), but did not alter cytosolic phosphoenolpyruvate carboxykinase (PCK1), resulting in a 2.4-fold greater PC:PCK1 ratio for FACW-supplemented cows compared with control. There was no evidence for a FACW effect on mRNA expression of propionyl-CoA carboxylase nor on mRNA expression or protein abundance of lactate dehydrogenase A or B. Cows supplemented with FACW had lower plasma urea nitrogen compared with control. Plasma l-lactate was greater for FACW-supplemented cows compared with control at 2 h before feeding time at 21 DIM. There was no evidence for altered expression of IL1B or IL10, or blood biomarkers related to liver function and damage. Greater glucose-6-phosphatase and PC gene expression, together with greater blood glucose and similar milk lactose output, suggests that FACW increased the supply of glucose precursors, resulting in greater gluconeogenesis between 3 and 14 DIM. Greater hepatic PC:PCK1 ratio, together with previously reported decreased plasma ß-hydroxybutyrate and the marginal evidence for lower liver triglyceride content at 14 DIM, suggests greater hepatic capacity for complete oxidation of fatty acids in FACW-supplemented cows compared with control. Overall, improvements in metabolite profile and feed efficiency observed with postpartum supplementation of FACW may be attributed to increased gluconeogenic and anaplerotic precursors, most likely propionate, due to modulated rumen fermentation.

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.

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.

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.

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.

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase.

Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1 mol of formate into 1 mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86 mM glucose and produced 172.38 mM succinate, 16.16 mM formate and 4.42 mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43 mM glucose and produced 171.80 mM succinate, 1mM formate and 5.78 mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40 g/L/h, 1g/L/h, and 17 mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2 g/L/h, 2 g/L/h, 0-3 mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal.

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.