Enzymes Of Gluconeogenesis Research Articles

Lr1-mediated leaf rust resistance pathways of transgenic wheat lines revealed by a gene expression study using the Affymetrix GeneChip® Wheat Genome Array

The wheat gene Lr1 imparts resistance to certain races of the leaf rust pathogen Puccinia triticina. However, the biological basis underlying the complex resistance mechanisms remains largely unknown. Gene expression profiling analyses using the Affymetrix GeneChip® Wheat Genome Array were performed with six independent Lr1 transgenic lines sampled before inoculation (0 h) and at 6 and 24 h post-inoculation (HPI) with the avirulent P. triticina race CCDS. For the 6- and 24-HPI time points, 3,384 probe sets corresponding to 2,976 transcripts were expressed differentially compared with the plants before inoculation. Genes involved in defence signalling are triggered as early as 6 HPI and include COI1, the primary component of jasmonic acid signalling. By 24 HPI, a shift in plant cellular metabolism, namely carbon conservation, is notable. The enzymes of glycolysis and gluconeogenesis are down-regulated; however, enzymes linked to alternative carbon sequestration processes such as the glyoxylate cycle are up-regulated. Also at 24 HPI, secondary signalling molecules belonging to the various hormone signalling pathways were triggered. These microarray data suggest that the Lr1-mediated leaf rust resistance is achieved by early onset of defence signalling followed by a coordinated interplay of metabolism alteration and elicitation of defence responses.

Effect of Simulated Microgravity on the Activity of Regulatory Enzymes of Glycolysis and Gluconeogenesis in Mice Liver

Gravity supports all the life activities present on earth. Microgravity environments have effect on the biological functions and physiological status of an individual. The present study was undertaken to investigate the effect of simulated microgravity on important regulatory enzymes of carbohydrate metabolism in liver using HLS mice model. Following hind limb unloading of mice for 11 days the animal’s average body weights were found to be not different, while the liver weights were decreased and found to be significantly different (p < 0.05) from control mice. Further, in liver the specific activity of hexokinase enzyme was reduced (p < 0.02) and the phosphoenolpyruvate carboxykinase activity was significantly increased in simulated microgravity subjected mice compared to control (p < 0.003). Immunoblot analysis show decreased phosphofructokinase-2 activity in HLS mice compared to control. Liver lactate dehydrogenase activity significantly reduced in simulated microgravity subjected mice (p < 0.005). Thus in our study the rodents have adapted to simulated microgravity conditions, with decreased glycolysis and increased gluconeogenesis in liver and reciprocally regulated.

Effect of fishmeal replacement by soy protein concentrate with taurine supplementation on hepatic intermediary metabolism and antioxidant status of totoaba juveniles (Totoaba macdonaldi)

The effect of dietary incorporation of soy protein concentrate (SPC) and the concomitant supplementation with taurine on hepatic intermediary metabolism and antioxidant status of totoaba (Totoaba macdonaldi) juveniles was assessed. Four isoproteic and isolipidic diets were formulated containing either 30 or 60% of SPC (diets SP30 and SP60), supplemented or not with 1% of taurine (diets SP30T and SP60T). A fish meal (FM) based diet, without SPC and taurine supplementation, was used as a control. Triplicate groups of 32 totoaba juveniles (average body mass=7.5g) were fed these diets over 45days. Results revealed that dietary FM replacement by SPC depressed the overall intermediary metabolism. Activity of key enzymes of amino acid catabolism and gluconeogenesis was significantly reduced and a trend to reduce glycolysis and glucose-6-phosphate dehydrogenase activity was observed. The incorporation of the highest level of SPC also significantly increased hepatic lipid peroxidation and the activity of superoxide dismutase. Concomitant taurine supplementation restored the activity of amino acid catabolic and gluconeogenic enzymes and hexokinase to levels similar of those of the control diet. Taurine supplementation also led to a significant increase of glucose-6-phosphate dehydrogenase and catalase activity, as well as to a significant reduction of liver lipid peroxidation. These results suggest that taurine may play an important metabolic modulation action on totoaba fed SPC based diets, contributing to the enhancement of the overall metabolism and to the reduction of liver oxidative damage.

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

BackgroundFructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. MethodsFour types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. ResultsThe kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. ConclusionsBinding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. General significanceMimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.

Immune and production responses of dairy cows to postruminal supplementation with phytonutrients

This study investigated the effect of phytonutrients (PN) supplied postruminally on nutrient utilization, gut microbial ecology, immune response, and productivity of lactating dairy cows. Eight ruminally cannulated Holstein cows were used in a replicated 4×4 Latin square. Experimental periods lasted 23d, including 14-d washout and 9-d treatment periods. Treatments were control (no PN) and daily doses of 2g/cow of either curcuma oleoresin (curcumin), garlic extract (garlic), or capsicum oleoresin (capsicum). Phytonutrients were pulse-dosed into the abomasum of the cows, through the rumen cannula, 2h after feeding during the last 9d of each experimental period. Dry matter intake was not affected by PN, although it tended to be lower for the garlic treatment compared with the control. Milk yield was decreased (2.2kg/d) by capsicum treatment compared with the control. Feed efficiency, milk composition, milk fat and protein yields, milk N efficiency, and 4.0% fat-corrected milk yield were not affected by treatment. Rumen fermentation variables, apparent total-tract digestibility of nutrients, N excretion with feces and urine, and diversity of fecal bacteria were also not affected by treatment. Phytonutrients had no effect on blood chemistry, but the relative proportion of lymphocytes was increased by the capsicum treatment compared with the control. All PN increased the proportion of total CD4+ cells and total CD4+ cells that co-expressed the activation status signal and CD25 in blood. The percentage of peripheral blood mononuclear cells (PBMC) that proliferated in response to concanavalin A and viability of PBMC were not affected by treatment. Cytokine production by PBMC was not different between control and PN. Expression of mRNA in liver for key enzymes in gluconeogenesis, fatty acid oxidation, and response to reactive oxygen species were not affected by treatment. No difference was observed due to treatment in the oxygen radical absorbance capacity of blood plasma but, compared with the control, garlic treatment increased 8-isoprostane levels. Overall, the PN used in this study had subtle or no effects on blood cells and blood chemistry, nutrient digestibility, and fecal bacterial diversity, but appeared to have an immune-stimulatory effect by activating and inducing the expansion of CD4 cells in dairy cows. Capsicum treatment decreased milk yield, but this and other effects observed in this study should be interpreted with caution because of the short duration of treatment.

Cynara Scolymus Leaf Extract Stimulates Adipogenesis in 3T3-L1 Preadipocytes and Suppresses Hepatic Glucose Output in H4IIE Hepatocytes

Insulin resistance is the hallmark of type 2 diabetes and is characterized by ectopic fat accumulation in insulin-sensitive tissues, including liver and skeletal muscle. Artichoke (Cynara scolymus) is 1 of the most commonly consumed foods around the world. Leaf extract of C. scolymus is reported to have important therapeutic activities. However, few studies have assessed the possible antidiabetic effects of artichoke extracts. In this study, we investigated the effects of artichoke leaf extract on adipogenesis in 3T3-L1 cells and on glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes. Preadipocytes were incubated with 25 μg/ mL of the leaf extract and lipid accumulation was assessed by AdipoRed assay. Rosiglitazone (10 μM), an oral hypoglycemic thiazolidinedione PPAR-γ agonist, was used as a positive control. Hepatic insulin resistance results in increased hepatic glucose output, the main factor behind fasting hyperglycemia in type 2 diabetes. G6Pase is the rate-limiting enzyme of gluconeogenesis, hence its inhibition reduces hepatic glucose production. We tested the effect of artichoke leaf extract (25 μg/mL) on the production of glucose by H4IIE, insulin (100 nM) being used as the positive control. Our results show that C. scolymus leaf extract significantly stimulated adipogenesis in 3T3-L1 adipocytes by 4 fold, as compared to 7.8-fold increase by rosiglitazone. The artichoke extract also inhibited G6Pase activity by 30% in H4IIE hepatocytes, as compared to 60% for insulin. These findings support the antidiabetic potential of this plant product.

Crystal Structures of Human Muscle Fructose-1,6-Bisphosphatase: Novel Quaternary States, Enhanced AMP Affinity, and Allosteric Signal Transmission Pathway

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, is subject to metabolic regulation. The human muscle isozyme is significantly more sensitive towards the allosteric inhibitor, AMP, than the liver isoform. Here we report crystal structures and kinetic studies for wild-type human muscle Fru-1,6-Pase, the AMP-bound (1.6 Å), and product-bound complexes of the Q32R mutant, which was firstly introduced by an error in the cloning. Our high-resolution structure reveals for the first time that the higher sensitivity of the muscle isozyme towards AMP originates from an additional water-mediated, H-bonded network established between AMP and the binding pocket. Also present in our structures are a metaphosphate molecule, alternate conformations of Glu97 coordinating Mg2+, and possible metal migration during catalysis. Although the individual subunit is similar to previously reported Fru-1,6-Pase structures, the tetrameric assembly of all these structures deviates from the canonical R- or T-states, representing novel tetrameric assemblies. Intriguingly, the concentration of AMP required for 50% inhibition of the Q32R mutant is increased 19-fold, and the cooperativity of both AMP and Mg2+ is abolished or decreased. These structures demonstrate the Q32R mutation affects the conformations of both N-terminal residues and the dynamic loop 52–72. Also importantly, structural comparison indicates that this mutation in helix α2 is detrimental to the R-to-T conversion as evidenced by the absence of quaternary structural changes upon AMP binding, providing direct evidence for the critical role of helix α2 in the allosteric signal transduction.

Expression of Phosphoenolpyruvate Carboxykinase Isoforms in The Mammary Gland of Dairy Goats is Regulated by Insulin Status

Phosphoenolpyruvate carboxykinase (PEPCK) isoforms (c, cytosolic; m, mitochondria) are expressed in the liver and mammary gland. PEPCK-c is a rate-controlling enzyme for gluconeogenesis and glyceroneogenesis; its activity is decreased by insulin. PEPCK-m expression is constitutive and functions to channel lactate toward gluconeogenesis. We hypothesized that the increase in milk protein but decrease in milk lactose and fat when a hyperinsulinemic-euglycemic clamp (HIEC) is applied to dairy goats is due to decreased expression of mammary PEPCK-c mRNA. Late lactation goats (n = 4; 150 ± 30 days in milk) were subjected to saline infusion and HIEC (104 µg insulin/h) for 4-day periods in a 2 × 2 crossover design. On day 4 of each period, a mammary biopsy (~1 g) was taken from an udder half to determine expres- sion of PEPCK-m and PEPCK-c mRNA by real-time RT-PCR. Plasma insulin increased 3.5-fold (P< 0.002) due to the HIEC and euglycemia was maintained. The HIEC decreased (P< 0.0001) dry matter intake (28%) and milk yield (26%). While milk fat content was not affected, HIEC increased (P< 0.001) milk protein content (2.82% vs. 3.09%) but decreased (P< 0.001) milk lactose content (4.22% vs. 4.03%). Expression of PEPCK-m mRNA was 9-fold higher (P< 0.004) than that of PEPCK-c. The HIEC decreased (P< 0.03) PEPCK-c mRNA 7-fold but tended to increase (P< 0.236) PEPCK-m mRNA 1.3-fold. These results demonstrate that insulin regulates mRNA expression of mammary PEPCK isoforms, and this may underlie the changes in milk-component synthesis observed when a HIEC is applied.

The Arabidopsis METACASPASE9 Degradome

Metacaspases are distant relatives of the metazoan caspases, found in plants, fungi, and protists. However, in contrast with caspases, information about the physiological substrates of metacaspases is still scarce. By means of N-terminal combined fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in young seedlings of Arabidopsis thaliana on the proteome-wide level, providing additional insight into MC9 cleavage specificity and revealing a previously unknown preference for acidic residues at the substrate prime site position P1'. The functionalities of the identified MC9 substrates hinted at metacaspase functions other than those related to cell death. These results allowed us to resolve the substrate specificity of MC9 in more detail and indicated that the activity of phosphoenolpyruvate carboxykinase 1 (AT4G37870), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis.

MicroRNA 33 Regulates Glucose Metabolism

Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small noncoding RNAs (microRNAs [miRNAs]) in the posttranscriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a and miR33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression, leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression of PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.

GCN2 regulates the CCAAT enhancer binding protein beta and hepatic gluconeogenesis

Mice deficient for general control nondepressible-2 (Gcn2) either globally or specifically in the liver display reduced capacity to maintain glucose homeostasis during fasting, suggesting the hypothesis that GCN2 may regulate gluconeogenesis (GNG), which normally plays a key role maintaining peripheral glucose homeostasis. Gcn2-deficient mice exhibit normal insulin sensitivity and plasma insulin but show reduced GNG when administered pyruvate, a gluconeogenic substrate. The basal expression of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme in GNG, is abnormally elevated in Gcn2 knockout (KO) mice in the fed state but fails to be further induced during fasting. The level of tricarboxylic acid cycle intermediates, including malate and oxaloacetate, and the NADH-to-NAD(+) ratio are perturbed in the liver of Gcn2 KO mice either in the fed or fasted state, which may directly impinge upon GNG. Additionally, the expression of the CCAAT enhancer-binding protein-β (C/EBPβ) in the liver fails to be induced in Gcn2 KO mice after 24 h fasting, and the liver-specific Cebpβ KO mice show reduced fasting GNG similar to that seen in Gcn2-deficient mice. Our study demonstrates that GCN2 is important in maintaining GNG in the liver, which is likely to be mediated through regulation of C/EBPβ.

Gene expression under chronic heat stress in populations of the mustard hill coral (Porites astreoides) from different thermal environments

Recent evidence suggests that corals can acclimatize or adapt to local stress factors through differential regulation of their gene expression. Profiling gene expression in corals from diverse environments can elucidate the physiological processes that may be responsible for maximizing coral fitness in their natural habitat and lead to a better understanding of the coral's capacity to survive the effects of global climate change. In an accompanying paper, we show that Porites astreoides from thermally different reef habitats exhibit distinct physiological responses when exposed to 6weeks of chronic temperature stress in a common garden experiment. Here, we describe expression profiles obtained from the same corals for a panel of 9 previously reported and 10 novel candidate stress response genes identified in a pilot RNA-Seq experiment. The strongest expression change was observed in a novel candidate gene potentially involved in calcification, SLC26, a member of the solute carrier family 26 anion exchangers, which was down-regulated by 92-fold in bleached corals relative to controls. The most notable signature of divergence between coral populations was constitutive up-regulation of metabolic genes in corals from the warmer inshore location, including the gluconeogenesis enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase and the lipid beta-oxidation enzyme acyl-CoA dehydrogenase. Our observations highlight several molecular pathways that were not previously implicated in the coral stress response and suggest that host management of energy budgets might play an adaptive role in holobiont thermotolerance.

The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases

The thermotolerant Gram-positive methylotroph Bacillus methanolicus is able to grow with methanol, glucose or mannitol as a sole carbon and energy source. Fructose 1,6-bisphosphate aldolase (FBA), a key enzyme of glycolysis and gluconeogenesis, is encoded in the genome of B. methanolicus by two putative fba genes, the chromosomally located fba(C) and fba(P) on the naturally occurring plasmid pBM19. Their amino acid sequences share 75 % identity and suggest a classification as class II aldolases. Both enzymes were purified from recombinant Escherichia coli and were found to be active as homotetramers. Both enzymes were activated by either manganese or cobalt ions, and inhibited by ADP, ATP and EDTA. The kinetic parameters allowed us to distinguish the chromosomally encoded FBA(C) from the plasmid encoded FBA(P), since FBA(C) showed higher affinity towards fructose 1,6-bisphosphate (Km of 0.16±0.01 mM as compared to 2±0.08 mM) as well as higher glycolytic catalytic efficiency (31.3 as compared to 0.8 s(-1) mM(-1)) than FBA(P). However, FBA(P) exhibited a higher catalytic efficiency in gluconeogenesis (50.4 as compared to 1.4 s(-1) mM(-1) with dihydroxyacetone phosphate and 4 as compared to 0.4 s(-1) mM(-1) with glyceraldehyde 3-phosphate as limiting substrate). The aldolase-negative Corynebacterium glutamicum mutant Δfda could be complemented with both FBA genes from B. methanolicus. Based on the kinetic data, we propose that FBA(C) acts as major aldolase in glycolysis, whereas FBA(P) acts as major aldolase in gluconeogenesis in B. methanolicus.

Cooperative Anti-Diabetic Effects of Deoxynojirimycin-Polysaccharide by Inhibiting Glucose Absorption and Modulating Glucose Metabolism in Streptozotocin-Induced Diabetic Mice

We had previously shown that deoxynojirimycin-polysaccharide mixture (DPM) not only decreased blood glucose but also reversed the damage to pancreatic β-cells in diabetic mice, and that the anti-hyperglycemic efficacy of this combination was better than that of 1-deoxynojirimycin (DNJ) or polysachharide alone. However, the mechanisms behind these effects were not fully understood. The present study aimed to evaluate the therapeutic effects of DPM on streptozotocin (STZ)-induced diabetic symptoms and their potential mechanisms. Diabetic mice were treated with DPM (150 mg/kg body weight) for 90 days and continued to be fed without DPM for an additional 30 days. Strikingly, decrease of blood glucose levels was observed in all DPM treated diabetic mice, which persisted 30 days after cessation of DPM administration. Significant decrease of glycosylated hemoglobin and hepatic pyruvate concentrations, along with marked increase of serum insulin and hepatic glycogen levels were detected in DPM treated diabetic mice. Results of a labeled 13C6-glucose uptake assay indicated that DPM can restrain glucose absorption. Additionally, DPM down-regulated the mRNA and protein expression of jejunal Na+/glucose cotransporter, Na+/K+-ATPase and glucose transporter 2, and enhanced the activities as well as mRNA and protein levels of hepatic glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylas E1). Activity and expression of hepatic gluconeogenesis enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) were also found to be attenuated in diabetic mice treated with DPM. Purified enzyme activity assays verified that the increased activities of glucose glycolysis enzymes resulted not from their direct activation, but from the relative increase in protein expression. Importantly, our histopathological observations support the results of our biochemical analyses and validate the protective effects of DPM on STZ-induced damage to the pancreas. Thus, DPM has significant potential as a therapeutic agent against diabetes.

Duodenal–Jejunal Bypass Surgery Up-Regulates the Expression of the Hepatic Insulin Signaling Proteins and the Key Regulatory Enzymes of Intestinal Gluconeogenesis in Diabetic Goto–Kakizaki Rats

Duodenal-jejunal bypass (DJB), which is not routinely applied in metabolic surgery, is an effective surgical procedure in terms of type 2 diabetes mellitus resolution. However, the underlying mechanisms are still undefined. Our aim was to investigate the diabetic improvement by DJB and to explore the changes in hepatic insulin signaling proteins and regulatory enzymes of gluconeogenesis after DJB in a non-obese diabetic rat model. Sixteen adult male Goto-Kakizaki rats were randomly divided into DJB and sham-operated groups. The body weight, food intake, hormone levels, and glucose metabolism were measured. The levels of protein expression and phosphorylation of insulin receptor-beta (IR-β) and insulin receptor substrate 2 (IRS-2) were evaluated in the liver. We also detected the expression of key regulatory enzymes of gluconeogenesis [phosphoenoylpyruvate carboxykinase-1 (PCK1), glucose-6-phosphatase-alpha (G6Pase-α)] in small intestine and liver. DJB induced significant diabetic improvement with higher postprandial glucagons-like peptide 1, peptide YY, and insulin levels, but without weight loss. The DJB group exhibited increased expression and phosphorylation of IR-β and IRS-2 in liver, up-regulated the expression of PCK1 and G6Pase-α in small intestine, and down-regulated the expression of these enzymes in liver. DJB is effective in up-regulating the expression of the key proteins in the hepatic insulin signaling pathway and the key regulatory enzymes of intestinal gluconeogenesis and down-regulating the expression of the key regulatory enzymes of hepatic gluconeogenesis without weight loss. Our study helps to reveal the potential role of hepatic insulin signaling pathway and intestinal gluconeogenesis in ameliorating insulin resistance after metabolic surgery.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (−386/−374), non-perfect DR1 (−118/−106) and HNF4α-specific binding motif (H4-SBM) (−26/−14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis

Fructose-1,6-bisphosphatase (FBPase), a key regulatory enzyme of gluconeogenesis, plays an essential role in metabolism and development of most organisms. To the wealth of available knowledge about FBPase from Clonorchis sinensis (CsFBPase), in this study, the characteristics of CsFBPase and its potential role in pathogenesis of clonorchiasis were investigated. The Km value of CsFBPase was calculated to be 41.9 uM. The optimal temperature and pH of CsFBPase were 37 °C and pH 7.5-8.0, respectively. In addition, Mg(2+) or K(+) played a regulatory role in enzyme activity of CsFBPase. Both transcriptional and translational level of CsFBPase were higher in metacercariae (one of larva stages) than those in adult worm (P < 0.05). CsFBPase were observed to extensively express in the intestine, vitellaria and tegument of adult worms and ubiquitously in metacercariae. Moreover, CsFBPase was confirmed as a component of excretory/secretory products. Consequently, the translocation of CsFBPase could be detected on epithelial cells of bile duct in liver of C. sinensis infected rat. Recombinant CsFBPase can specifically bind to the membrane of human hepatic stellate cell line LX-2 by immunofluorescence analysis and stimulated proliferation and activation of LX-2 which demonstrated by Cell Counting Kit-8 and upregulation of key fibrosis-related factors, such as α-smooth muscle actin, collagen I and collagen III using qRT-PCR. Thus, we predicated that CsFBPase might be a multifunctional enzyme which played as both regulatory enzyme and virulence factor in pathogenesis of C. sinensis infection.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

1-deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice

We investigated the role of 1-deoxynojirimycin (DNJ) on glucose absorption and metabolism in normal and diabetic mice. Oral and intravenous glucose tolerance tests and labeled 13C6-glucose uptake assays suggested that DNJ inhibited intestinal glucose absorption in intestine. We also showed that DNJ down-regulated intestinal SGLT1, Na+/K+-ATP and GLUT2 mRNA and protein expression. Pretreatment with DNJ (50 mg/kg) increased the activity, mRNA and protein levels of hepatic glycolysis enzymes (GK, PFK, PK, PDE1) and decreased the expression of gluconeogenesis enzymes (PEPCK, G-6-Pase). Assays of protein expression in hepatic cells and in vitro tests with purified enzymes indicated that the increased activity of glucose glycolysis enzymes was resulted from the relative increase in protein expression, rather than from direct enzyme activation. These results suggest that DNJ inhibits intestinal glucose absorption and accelerates hepatic glucose metabolism by directly regulating the expression of proteins involved in glucose transport systems, glycolysis and gluconeogenesis enzymes.

Synthetic FXR Agonist GW4064 Prevents Diet-Induced Hepatic Steatosis and Insulin Resistance

To examine the effect of farnesoid X receptor (FXR) activation by its synthetic agonist, 3-[2-[2-Chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) on diet-induced obesity and hepatic steatosis. Fifteen week-old C57BL/6 mice fed with high-fat diet (HFD) or high-fat, high-cholesterol diet were treated by twice weekly injection of GW4064 (50mg/kg) intraperitoneally or DMSO (carrier solution) for 6weeks. Body weight, body composition and food intake were monitored weekly. Serum glucose and insulin levels and lipid content in the liver were measured at the end of study. Additionally, genes involved in lipogenesis, gluconeogenesis and inflammation were analyzed by real time PCR. CD36 protein level was detected by western blot. Activation of FXR by GW4064 suppressed weight gain in C57BL/6 mice fed with either HFD or high-fat and high-cholesterol diet. GW4064 treatment of mice significantly repressed diet-induced hepatic steatosis as evidenced by lower triglyceride and free fatty acid level in the liver. Analysis of genes involved in lipid metabolism showed GW4064 markedly reduced lipid transporter Cd36 gene expression without affecting expression of genes that are directly involved in lipogenesis. GW4064 treatment attenuated hepatic inflammation while having no effect on white adipose tissue. In addition, activation of FXR by GW4064 avoided diet-induced hyperinsulinemia and hyperglycemia through decreasing the transcript levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6pase), two key enzymes in gluconeogenesis. The results verify the important function of FXR in diet-induced obesity and suggest that FXR agonists are promising therapeutic agents for obesity-associated metabolic disorders.