Fructose Bisphosphatase Research Articles

2466 MicroRNA-451: A potential key player in the development of diabetic nephropathy in an insulin resistant mouse model

OBJECTIVES/SPECIFIC AIMS: MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA-451 may protect against DN and reduce renal gluconeogenesis in rodent models. MiRNA-451 is thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic-target of rapamycin (mTOR) in early DN. This study aimed to elucidate the role of miRNA-451 in the development and progression of DN. METHODS/STUDY POPULATION: To further elucidate the role of miRNA-451 in DN, we placed male insulin-resistant, TALLYHO/Jng mice on a high-fat diet (60% kCal). The mice were divided into 2 treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR-451-inhibitor or LNA-scrambled compound (2 mg/kg·bw; n=8/treatment). Mice were euthanized after 12 weeks (4 weeks sans injections) and kidneys, liver, pancreas and abdominal adipose tissue were harvested for analysis. RESULTS/ANTICIPATED RESULTS: Renal homogenate expression of miRNA-451 was drastically reduced in inhibitor-treated mice (~6-fold) in comparison with scramble-treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA-451 revealed a significant reduction in collagen IV (marker of epithelial integrity) in inhibitor-treated mice. In addition, metalloproteinase type 9 (MMP9, a known type IV collagenase), YWHAZ (a scaffolding protein and known target of miR-451), mTOR, and fructose bisphosphatase (FBP1, a rate-limiting gene in gluconeogenesis) were significantly increased in this group in comparison to scramble-treated mice. However, no differences were found in protein levels for glucose-6-phosphatase (G-6-Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate-limiting genes. MiRNA-451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA-inhibitor treated group (when compared with the scramble-treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA-inhibitor treated group when compared with scramble-treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson Trichrome scoring revealed a 59% increase in fibrosis in inhibitor-treated mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these findings support a potentially protective role of miRNA-451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA-451 signaling may be beneficial to reduce renal pathology associated with DN.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH2+) for CO2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway.

Renal impact of systemic inhibition of miRNA‐451 in a mouse model of insulin resistance

MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA‐451 may protect against DN and reduce renal gluconeogenesis in rodent models. It is hypothetically thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic‐target of rapamycin (mTOR) in early DN. To further elucidate the role of miRNA‐451 in DN, we placed male insulin‐resistant, TALLYHO/Jng mice on a high‐fat diet (60% kCal). The mice were divided into two treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR‐451‐inhibitor or LNA‐scrambled compound (2 mg/kg·bw; n = 8/treatment). Mice were euthanized after 12 weeks (4 weeks sans injections). Renal expression of miRNA‐451 was drastically reduced in inhibitor treated mice (~6‐fold) in comparison to scramble‐treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA‐451 revealed a significant reduction in collagen IV (marker of epithelial integrity) in inhibitor treated mice. In addition, metalloproteinase type 9 (MMP9, a known type IV collagenase), YWHAZ (a scaffolding protein and known target of miR‐451), mTOR, and fructose bisphosphatase (FBP1, a rate‐limiting gene in gluconeogenesis) were significantly increased in this group in comparison to scramble‐treated mice. However, no differences were found in protein levels for glucose‐6‐phosphatase (G‐6‐Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate‐limiting genes. MiRNA‐451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA‐inhibitor treated group (when compared to the scramble‐treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA‐inhibitor treated group when compared to scramble‐treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson's Trichrome scoring revealed a 59% increase in fibrosis in inhibitortreated mice. Overall, these studies support a potentially protective role of miRNA‐451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA‐451 signaling may be beneficial to reduce renal pathology associated with DN.Support or Funding InformationResearch reported in this abstract was supported by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award Number TL1TR001431 and the Georgetown University Department of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Responses to short‐term acid loading in proximal‐tubule‐select insulin/insulin‐like growth factor receptor dual knockout mice

Acidosis has been associated with increased renal gluconeogenesis, which in the absence of liver compensation, can lead to hyperglycemia. Acidosis leads to glutamine uptake in the proximal tubule (PT) to liberate bicarbonate and neutralize acids. The remaining carbon skeleton can be used for glucose production at this site. Whether insulin or the related hormone, insulin‐like‐growth‐factor, type 1 (IGF1), modulate these responses to acidosis is unclear. To address this, we bred mice with dual knockout (KO) of the insulin (Insr) and the Igf1r receptors from PT by Cre‐lox recombination using a γ‐glutamyl transferase promoter. KO mice had on average 40% lower expression of both Igf1r and Insr in cortex homogenates. To address the effects of acidosis, male (M) and female (F) wild‐type (WT, Cre negative) and KO (Cre positive) mice were provided ad libitum access to normal drinking water or a water‐based solution containing 1.5% NH4Cl for 7 days (n = 6/treatment/genotype/sex). Fasting (18‐hr) blood glucose was determined in a subset of mice after one week of control then acid treatment. MKO mice showed the greatest mean rise in fasting blood glucose with acid (mg/dl, delta acid‐control): −1.67 (MWT); +15.75 (MKO); −6.40 (FWT); +4.08 (FKO). Urine (24 hr) was collected in metabolic cages on days 5–6. Acid water led to a 30–64% reduction in urine volume in all groups except MWT (ml, delta acid‐control): +0.49 (MWT); −0.54 (MKO); −0.90 (FWT); −1.50 (FKO). Blood chemistry revealed greater sensitivity to acid loading in the male KO. Mean deltas (acid‐control) for blood bicarbonate, base excess, and anion gap, respectively were (mM): −5.73, −7.8, +1.24 (MWT); −8.86, −11.30, +4.67 (MKO); −7.35, −10.36, +4.74 (FWT); −1.67, −1.92, −0.6 (FKO). Western blotting for 3 rate‐limiting enzymes in gluconeogenesis conducted on the cortex from male mice revealed a significant rise in glucose‐6‐phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK), and fructose bisphosphatase (FBP1) in the acid drinking WT males (16–34%). In contrast, MKO mice had a significant reduction in G6P (22%) with acid water; however, FBP1 was increased by acid drinking to a greater extent (60%) in the KO, (as compared to WT, significant interaction). Overall our studies reveal a role for PT‐associated Insr and/or Igf1r signaling in regulating PT responses to an acid load and glucose homeostasis.Support or Funding InformationGeorgetown University Medical CenterGeorgetown/Howard CTSA awardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fructose bisphosphatase 2 overexpression increases glucose uptake in skeletal muscle.

Skeletal muscle is a major tissue for glucose metabolism and can store glucose as glycogen, convert glucose to lactate via glycolysis and fully oxidise glucose to CO2 Muscle has a limited capacity for gluconeogenesis but can convert lactate and alanine to glycogen. Gluconeogenesis requires FBP2, a muscle-specific form of fructose bisphosphatase that converts fructose-1,6-bisphosphate (F-1,6-bisP) to fructose-6-phosphate (F-6-P) opposing the activity of the ATP-consuming enzyme phosphofructokinase (PFK). In mammalian muscle, the activity of PFK is normally 100 times higher than FBP2 and therefore energy wasting cycling between PFK and FBP2 is low. In an attempt to increase substrate cycling between F-6-P and F-1,6-bisP and alter glucose metabolism, we overexpressed FBP2 using a muscle-specific adeno-associated virus (AAV-tMCK-FBP2). AAV was injected into the right tibialis muscle of rats, while the control contralateral left tibialis received a saline injection. Rats were fed a chow or 45% fat diet (HFD) for 5 weeks after which, hyperinsulinaemic-euglycaemic clamps were performed. Infection of the right tibialis with AAV-tMCK-FBP2 increased FBP2 activity 10 fold on average in chow and HFD rats (P < 0.0001). Overexpression of FBP2 significantly increased insulin-stimulated glucose uptake in tibialis of chow animals (control 14.3 ± 1.7; FBP2 17.6 ± 1.6 µmol/min/100 g) and HFD animals (control 9.6 ± 1.1; FBP2 11.2 ± 1.1µmol/min/100 g). The results suggest that increasing the capacity for cycling between F-1,6-bisP and F-6-P can increase the metabolism of glucose by introducing a futile cycle in muscle, but this increase is not sufficient to overcome muscle insulin resistance.

In ovo feeding of L-arginine alters energy metabolism in post-hatch broilers.

This study aimed to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on energy metabolism in post-hatch broilers. A total of 720 eggs was randomly assigned to 3 treatments: 1) non-injected control group, 2) 0.75% NaCl diluent-injected control group, and 3) 1.0% Arg solution-injected group. At 17.5 d of incubation, 0.6mL of each solution was injected into the amniotic fluid of each egg of injected groups. After hatching, 80 male chicks were randomly assigned to each treatment group with 8 replicates per group. The results showed that IOF of Arg increased glycogen and glucose concentrations in the liver and pectoral muscle of broilers at hatch (P <0.05). The plasma glucose and insulin levels were higher in the Arg group than in the non-injected and diluent-injected control groups (P <0.05). Meanwhile, IOF of Arg enhanced the hepatic glucose-6-phosphatase (G6P) activity at hatch (P <0.05). There was no difference in hexokinase (HK) or phosphofructokinase (PFK) enzyme activities in the pectoral muscle in all groups. Further, IOF of Arg increased the phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FBP) mRNA expressions at hatch (P <0.05). In addition, broilers in the Arg group had a higher mRNA expression of glycogen synthase and a lower expression of glycogen phosphorylase in the liver and pectoral muscles than in the non-injected controls at hatch (P <0.05). In conclusion, IOF of Arg solution enhanced liver and pectoral muscle energy reserves at hatch, which might be considered as an effective strategy for regulating early energy metabolism in broilers.

Effects of dietary carbohydrate sources on growth, digestive enzyme activity, gene expression of hepatic GLUTs and key enzymes involved in glycolysis-gluconeogenesis of giant grouper Epinephelus lanceolatus larvae

This study aimed to evaluate effects of dietary carbohydrate sources on growth, digestive enzyme activity, gene expression of hepatic glucose transporters (GLUTs) and key enzymes involved in glycolysis-gluconeogenesis in giant grouper Epinephelus lanceolatus larvae through a 21-day growth trial. Four isonitrogenous (50%) and isolipidic (15.4%) experimental diets, including a cellulose control diet and three diets with glucose, maltose or maize starch, were formulated. Dietary CHO level was 18%. The 29 DPH (days post hatching) grouper larvae (initial average weight: 0.078±0.0018g) were cultured in small floating cages (L 120cm×W 70cm×H 50cm) at a density of 80 fish per cage. Experimental cages were labeled and located in three connective 6-m3 indoor concrete tanks (L 3m×W 3m×H 2m) with 4 cages occurring in each tank.Results showed that at 50 DPH, fish fed with maize starch diet had significantly higher weight gain (WG) and thermal growth coefficient (TGC) than fish fed with glucose, maltose or cellulose. Fish fed with glucose had the lowest WG among all experimental treatments. Fish fed with maize starch or cellulose had significantly higher mRNA expression levels of hepatic fructose 1,6 bisphosphatase (FBP) gene compared to fish fed with glucose or maltose at 50 DPH. The highest mRNA expression level of hepatic glucose-6-phosphate dehydrogenase (G6PDH) gene was observed in fish fed with glucose at 50 DPH. Fish fed with maize starch had higher mRNA expression levels of hepatic pyruvate kinase (PK) and hexokinase (HK) genes than fish fed with cellulose, glucose and maltose at 50 DPH. The mRNA expression level of hepatic glucose transporter 4 (GLUT4) gene was significantly lower in fish fed with cellulose compared to that in fish fed with digestible CHOs at 50 DPH. There were no significant differences in mRNA expression levels of hepatic glucose transporter 1 (GLUT1) gene among all experimental fish at 50 DPH. In terms of digestive enzymes, activities of pancreatic trypsin and intestinal lipase increased during the development of fish larvae, but pancreatic α-amylase specific activity showed an opposite trend of variation as those of trypsin and lipase. During the development of experimental fish larvae from 29 DPH to 50 DPH, pancreatic trypsin specific activity in fish fed with glucose was significantly lower than that in fish fed with cellulose or maltose but did not differ from that in fish fed with maize starch. Intestinal lipase specific activity in fish fed with maize starch or maltose were significantly higher than those in fish fed with cellulose but did not differ from those in fish fed with glucose. Generally, giant grouper larvae fed maize starch had better growth than those fed cellulose, glucose or maltose; the mRNA expressions of hepatic HK, PK, FBP and G6PDH genes involved in glycolysis-gluconeogenesis were significantly affected by dietary CHO sources.

Description of carbon fixation pathway based on &amp;lt;italic&amp;gt;Skeletonema marinoi&amp;lt;/italic&amp;gt; transcriptome

Diatoms can fix approximately 1016 g CO2 into organic carbon every year, equivalent to roughly 20% of global primary production. They are the basis for important food webs, having considerable impacts on carbon cycle and biological carbon pump that draws down CO2 from the atmosphere to the ocean interior. Nevertheless, the K 1/2 of their ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is 40~60 μ mol L - 1, higher than the apparent photosynthetic K 1/2 (CO2) for ambient CO2. Growth and kinetic studies have shown that diatoms can avoid CO2 limitation through CO2 concentrating mechanisms. Recently the carbon fixation metabolism pathway in diatoms is further elucidated by the presence of a complete set of genes essential for this metabolic route in Thalassiosira pseudonana and Phaeodactylum tricornutum because of their entire availablegenome information. In this study, we used RNA-Seq technology to confirm the existence of necessary genes for plant-like C4 activity in Skeletonema marinoi , which is a complex biological trait that enables S. marinoi to either has high water-use efficiency or accumulate biomass at a fast rate. The carbon fixation metabolism pathway of S. marinoi involves 20 enzymes and 34 coding genes, including the complete key enzymes especially for C4 pathway, which are phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate orthophosphate dikinase (PPDK). Comparing with the homologous genes of carbon fixation metabolism pathway in T. pseudonana and P. tricornutum , these genes showed little divergence. Besides, the gene differential expression of S. marinoi in carbon fixation metabolism pathway was identified at growth stages using digital gene expression profiling. The results indicated the number of differential expression genes was 7 and 3 in C3 and C4 pathway, respectively. As for the differential expression of coding genes in growth stages, there were 8 and 10 coding genes in the stationary and decline phase, respectively versus to those in the exponential phase. The gene expression of fructose-1,6-bisphosphatase (FBP), fructose-bisphosphate aldolase (ALDO) and PPDK was changed significantly after the exponential phase. The FBP and ALDO are not only responsible for the carbon fixation metabolism pathway, but also as the key enzymes in the glycolytic metabolism pathway, which links closely with the flow direction of carbon and the synthesis of carbohydrates. The PPDK, a cardinal enzyme of the C4 pathway, catalyzing the regeneration of phosphoenol pyruvate(PEP), evolved as an adaptation to high light intensity, high temperature, low concentration of CO2 and dryness. Therefore, the formation of PEP through PPDK is considered to be very important in the C4 pathway. This study will contribute to analyze the gene regulation of key enzymes involved in the carbon metabolism of S. marinoi , explain the reason for huge amounts of primary production by diatom, and enhance our knowledge on the carbon fixation mechanism, which provides a new insight into understanding carbon biogeochemical cycle.

Maternal rumen-protected methionine supplementation and its effect on blood and liver biomarkers of energy metabolism, inflammation, and oxidative stress in neonatal Holstein calves

In nonruminants, nutrition during pregnancy can program offspring development, metabolism, and health in later life. Rumen-protected Met (RPM) supplementation during the prepartum period improves liver function and immune response in dairy cows. Our aim was to investigate the effects of RPM during late pregnancy on blood biomarkers (23 targets) and the liver transcriptome (24 genes) in neonatal calves from cows fed RPM at 0.08% of diet dry matter/d (MET) for the last 21 d before calving or controls (CON). Blood (n=12 calves per diet) was collected at birth before receiving colostrum (baseline), 24 h after receiving colostrum, 14, 28, and 50 d (post-weaning) of age. Liver was sampled (n=8 calves per diet) via biopsy on d 4, 14, 28, and 50 of age. Growth and health were not affected by maternal diet. The MET calves had greater overall plasma insulin concentration and lower glucose and ratios of glucose-to-insulin and fatty acids-to-insulin, indicating greater systemic insulin sensitivity. Lower concentration of reactive oxygen metabolites at 14 d of age along with a tendency for lower overall concentration of ceruloplasmin in MET calves indicated a lesser degree of stress. Greater expression on d 4 of fructose-bisphosphatase 1 (FBP1), phosphoenolpyruvate carboxykinase 1 (PCK1), and the facilitated bidirectional glucose transporter SLC2A2 in MET calves indicated alterations in gluconeogenesis and glucose uptake and release. The data agree with the greater expression of the glucocorticoid receptor (GR). Greater expression on d 4 of the insulin receptor (INSR) and insulin-responsive serine/threonine-protein kinase (AKT2) in MET calves indicated alterations in insulin signaling. In that context, the similar expression of sterol regulatory element-binding transcription factor 1 (SREBF1) in CON and MET during the preweaning period followed by the marked upregulation regardless of diet after weaning (d 50) support the idea of changes in hepatic insulin sensitivity during early postnatal life. Expression of carnitine palmitoyltransferase 1A (CPT1A) was overall greater and acyl-CoA oxidase 1 (ACOX1) was lower in MET calves, indicating alterations in fatty acid oxidation. Except forkhead box O1 (FOXO1), all genes changed in expression over time. Transcriptome results indicated that calves from MET-supplemented cows underwent a faster maturation of gluconeogenesis and fatty acid oxidation in the liver, which would be advantageous for adapting to the metabolic demands of extrauterine life.

A summary of molecular genetic findings in fructose-1,6-bisphosphatase deficiency with a focus on a common long-range deletion and the role of MLPA analysis.

BackgroundFructose-1,6-bisphosphatase deficiency is a rare inborn error of metabolism affecting gluconeogenesis with only sporadic reports on its molecular genetic basis.ResultsWe report our experience with mutation analysis in 14 patients (13 families) with fructose-1,6-bisphosphatase deficiency using conventional Sanger sequencing and multiplex ligation-dependent probe amplification analysis, and we provide a mutation update for the fructose bisphosphatase-1 gene (FBP1). Mutations were found on both chromosomes in all of our 14 patients including 5 novel mutations. Among the novel mutations is a 5412-bp deletion (c.-24-26_170 + 5192del) including the entire coding sequence of exon 2 of FBP1 that was repeatedly found in patients from Turkey and Armenia which may explain earlier poorly defined findings in patients from this area. This deletion can be detected with specific primers by generation of a junction fragment and by MLPA and SNP array assays. MLPA analysis was able to detect copy number variations in two further patients, one heterozygous for a deletion within exon 8, another heterozygous for a novel deletion of the entire FBP1 gene.ConclusionsBased on our update for the FBP1 gene, currently listing 35 mutations worldwide, and knowledge of PCR conditions that allow simple detection of a common FBP1 deletion in the Armenian and Turkish population, molecular genetic diagnosis has become easier in FBP1 deficiency. Furthermore, MLPA analysis may plays a useful role in patients with this disorder.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0415-1) contains supplementary material, which is available to authorized users.

Role of HIF-1 on phosphofructokinase and fructose 1, 6-bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei

HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and β subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.

Identification of the TP53-induced glycolysis and apoptosis regulator in various stages of colorectal cancer patients.

The TP53-induced glycolysis and apoptosis regulator (TIGAR) is a p53 target gene known to regulate glycolysis by acting as fructose bis-phosphatase (FBPase) and modulate reactive oxygen species. TIGAR expression has been implicated in oncogenesis and progression of several human cancers. However, TIGAR expression is not known in various stages of colorectal cancer (CRC). There is an increase in the colorectal cancer incidence in Saudi Arabia. We sought to analyze TIGAR expression in this ethnic group. The aim of this study was to investigate the TIGAR expression in colorectal cancer (CRC) patients from Saudi Arabia. Tissue microarray (TMA) was constructed from 22 matched colorectal tumor tissues and adjacent normal tissues. TIGAR expression was examined in TMA slide using immunohistochemistry. TIGAR mRNA was determined in 14 matched tumor tissue and adjacent normal tissue. TIGAR protein expression was also examined in CRC tumor tissues and cell lines. Statistical analyses (t-test) were applied to evaluate the significance of TIGAR expression. TIGAR mRNA level was upregulated significantly in stage II (p<0.01) and stage III (p<0.05) when compared to adjacent normal tissue. Immunohistochemical studies revealed that TIGAR expression was increased in colorectal cancer. Strong TIGAR positive staining was found in 68% (15/22) of the tumor samples with nuclear localization. TIGAR staining was found to be significantly increased in early stage (stage I and II) CRC (p<0.05) and late stage (stage III and IV) CRC (p<0.01). TIGAR protein was also found to be highly expressed in stage II and III colorectal cancer tissues and CRC cell lines. These findings indicate that TIGAR is highly expressed at the mRNA and protein levels in colorectal cancer with prominent nuclear localization. TIGAR expression may be used as a bio-marker for detection of colorectal cancer and can be used as a target for developing therapeutics for the treatment of colorectal cancer.

Homeostasis and the glycogen shunt explains aerobic ethanol production in yeast

Aerobic glycolysis in yeast and cancer cells produces pyruvate beyond oxidative needs, a paradox noted by Warburg almost a century ago. To address this question, we reanalyzed extensive measurements from (13)C magnetic resonance spectroscopy of yeast glycolysis and the coupled pathways of futile cycling and glycogen and trehalose synthesis (which we refer to as the glycogen shunt). When yeast are given a large glucose load under aerobic conditions, the fluxes of these pathways adapt to maintain homeostasis of glycolytic intermediates and ATP. The glycogen shunt uses glycolytic ATP to store glycolytic intermediates as glycogen and trehalose, generating pyruvate and ethanol as byproducts. This conclusion is supported by studies of yeast with a partial block in the glycogen shunt due to the cif mutation, which found that when challenged with glucose, the yeast cells accumulate glycolytic intermediates and ATP, which ultimately leads to cell death. The control of the relative fluxes, which is critical to maintain homeostasis, is most likely exerted by the enzymes pyruvate kinase and fructose bisphosphatase. The kinetic properties of yeast PK and mammalian PKM2, the isoform found in cancer, are similar, suggesting that the same mechanism may exist in cancer cells, which, under these conditions, could explain their excess lactate generation. The general principle that homeostasis of metabolite and ATP concentrations is a critical requirement for metabolic function suggests that enzymes and pathways that perform this critical role could be effective drug targets in cancer and other diseases.

Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis.

The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway.

Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation.

Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.Results: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.Discussion: We identified a unique HMOX1down and FBPup phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

Expression of fructose 1,6-bisphosphatase and phosphofructokinase is induced in hepatopancreas of the white shrimp Litopenaeus vannamei by hypoxia

Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.

Glucose metabolism and gene expression in juvenile zebrafish (Danio rerio) challenged with a high carbohydrate diet: effects of an acute glucose stimulus during late embryonic life.

Knowledge on the role of early nutritional stimuli as triggers of metabolic pathways in fish is extremely scarce. The objective of the present study was to assess the long-term effects of glucose injection in the yolk (early stimulus) on carbohydrate metabolism and gene regulation in zebrafish juveniles challenged with a high-carbohydrate low-protein (HC) diet. Eggs were microinjected at 1 d post-fertilisation (dpf) with either glucose (2 M) or saline solutions. Up to 25 dpf, fish were fed a low-carbohydrate high-protein (LC) control diet, which was followed by a challenge with the HC diet. Survival and growth of 35 dpf juveniles were not affected by injection or the HC diet. Glucose stimulus induced some long-term metabolic changes in the juveniles, as shown by the altered expression of genes involved in glucose metabolism. On glycolysis, the expression levels of hexokinase 1 (HK1) and phosphofructokinase-6 (6PFK) were up-regulated in the visceral and muscle tissues, respectively, of juveniles exposed to the glucose stimulus, indicating a possible improvement in glucose oxidation. On gluconeogenesis, the inhibition of the expression levels of PEPCK in fish injected with glucose suggested lower production of hepatic glucose. Unexpectedly, fructose-1,6-bisphosphatase (FBP) expression was induced and 6PFK expression reduced by glucose stimulus, leaving the possibility of a specific regulation of the FBP-6PFK metabolic cycle. Glucose metabolism in juveniles was estimated using a [¹⁴C]glucose tracer; fish previously exposed to the stimulus showed lower retention of [¹⁴C]glucose in visceral tissue (but not in muscle tissue) and, accordingly, higher glucose catabolism, in comparison with the saline group. Globally, our data suggest that glucose stimulus at embryo stage has the potential to alter particular steps of glucose metabolism in zebrafish juveniles.

Potassium diformate influences gene expression of GH/IGF-I axis and glucose homeostasis in weaning piglets

The objective of this study was to determine the effects of potassium diformate (KDF) on growth performance of weaning piglets and genes expression related with growth axis and hepatic gluconeogenesis. A total of 180 piglets weaned at 28d of age were allocated randomly into two groups, with 6 pens in each group and 15 piglets in each pen. Piglets in the control group were fed basal diet, whereas the KDF-treated group was fed basal diet supplemented with 10g/kg KDF. After 5 weeks feeding, KDF-treated piglets showed higher average daily feed intake (ADFI) and average daily bodyweight gain (ADG) than those of the control (P<0.05), whereas the feed conversion ratio (FCR) was lower than that of the control (P=0.02). The apparent digestibility of dry matter and crude protein of the KDF-treated group were higher than those of the control group (P<0.01). KDF treatment did not change the blood indices level of insulin-like growth factor-I (IGF-I), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC), triglyceride (TG) and blood glucose (BG) in plasma. KDF treatment did not influence the mRNA expression of growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and the levels of growth hormone receptor (GHR) and IGF-I receptor (IGF-IR) mRNA in pituitary. Whereas, it increased the abundance of GH mRNA in pituitary, and the mRNA of GHR, IGF-I, IGF-IR, glucose-6-phosphatase (G6PC), fructose-1,6-bisphosphatase (FBP) and phosphoenolpyruvate carboxykinase (PCK) in liver (P<0.05). Furthermore, KDF treatment increased the concentration of IGF-I peptide in liver (P=0.000). These results suggest that, the beneficial effect of KDF on the growth performance may relate to the regulation of the GH axis and glucose homeostasis in piglets.

Systems-Level Analysis of Nitrogen Starvation-Induced Modifications of Carbon Metabolism in a Chlamydomonas reinhardtii Starchless Mutant

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Transient pseudo-hypertriglyceridemia: a useful biochemical marker of fructose-1,6-bisphosphatase deficiency

Fructose-1,6-bisphosphatase (FBP) deficiency is an autosomal-recessive disorder of gluconeogenesis resulting from mutations within the FBP1 gene. During periods of trivial illness, individuals with FBP deficiency may develop ketotic hypoglycemia, metabolic acidosis, lactic acidemia, and an increased anion gap. Although detection of urinary excretion of glycerol by urine organic acid analysis has been previously described, the presence of transient pseudo-hypertriglyceridemia in serum during metabolic decompensation has not been reported before. This study describes four consanguineous Pakistani families, in which four patients were diagnosed with FBP deficiency. All showed transient pseudo-hypertriglyceridemia during the acute phase of metabolic decompensation, which resolved in a metabolically stable phase. Mutations in the FBP1 gene have been described from various ethnicities, but there is very limited literature available for the Pakistani population. This study also describes one novel mutation in the FBP1 gene which seems to be prevalent in Pakistani-Indian patients. As a result of this study, transient pseudo-hypertriglyceridemia should be added to glyceroluria, ketotic hypoglycemia, metabolic acidosis, and lactic acidosis as a useful biochemical marker of FBP deficiency.