Enzymes Of Gluconeogenesis Research Articles

SCP4 Promotes Gluconeogenesis Through FoxO1/3a Dephosphorylation.

FoxO1 and FoxO3a (collectively FoxO1/3a) proteins regulate a wide array of cellular processes, including hepatic gluconeogenesis. Phosphorylation of FoxO1/3a is a key event that determines its subcellular location and transcriptional activity. During glucose synthesis, the activity of FoxO1/3a is negatively regulated by Akt-mediated phosphorylation, which leads to the cytoplasmic retention of FoxO1/3a. However, the nuclear phosphatase that directly regulates FoxO1/3a remains to be identified. In this study, we discovered a nuclear phosphatase, SCP4/CTDSPL2 (SCP4), that dephosphorylated FoxO1/3a and promoted FoxO1/3a transcription activity. We found that SCP4 enhanced the transcription of FoxO1/3a target genes encoding PEPCK1 and G6PC, key enzymes in hepatic gluconeogenesis. Ectopic expression of SCP4 increased, while knockdown of SCP4 inhibited, glucose production. Moreover, we demonstrated that gene ablation of SCP4 led to hypoglycemia in neonatal mice. Consistent with the positive role of SCP4 in gluconeogenesis, expression of SCP4 was regulated under pathophysiological conditions. SCP4 expression was induced by glucose deprivation in vitro and in vivo and was elevated in obese mice caused by genetic (Avy) and dietary (high-fat) changes. Thus, our findings provided experimental evidence that SCP4 regulates hepatic gluconeogenesis and could serve as a potential target for the prevention and treatment of diet-induced glucose intolerance and type 2 diabetes.

Fructose-1,6-bisphosphatase Inhibits ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking IQGAP1-MAPK Interaction.

Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1-MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1-IQGAP1-ERK1/2 signaling axis can be targeted for effective treatment of PDAC. Cancer Res; 77(16); 4328-41. ©2017 AACR.

Role of AMPD2 in impaired glucose tolerance induced by high fructose diet

A high intake of products containing fructose is known to mediate insulin resistance. In the liver, AMPD2, an isoform of AMPD, has important glucose metabolic homeostasis functions including maintenance of AMP-activated protein kinase (AMPK). We speculated that AMPD2 induces impaired glucose tolerance in individuals who consume a high-fructose diet. We gave either a normal-chow (NCD) or high-fructose (HFrD) diet for 40days to 8-week-old male wild-type (WT) and Ampd2−/− homozygote (A2−/−) C57BL/6 mice. A glucose tolerance test (GTT) and pyruvate tolerance test (PTT) were used to evaluate glucose metabolism. In addition, gluconeogenesis and glycolysis enzymes, and AMPK phosphorylation in the liver were investigated. With consumption of the HFrD, A2−/− mice showed enhanced glucose tolerance in GTT and PTT results as compared to the WT mice, which were independent of changes in body weight. Also, the levels of phosphoenolpyruvate carboxy kinase and glucose-6-phosphatase (hepatic gluconeogenic enzymes) were significantly reduced in A2−/− as compared to WT mice. The hepatic glycolytic enzymes glucokinase, phosphofructokinase, and pyruvate kinase were also examined, though there were no significant differences between genotypes in regard to both mRNA expression and protein expression under HFrD. Surprisingly, hepatic AMPK phosphorylation resulted in no changes in the A2−/− as compared to WT mice under these conditions. Our results indicated that Ampd2–deficient mice are protected from high fructose diet-induced glycemic dysregulation, mainly because of gluconeogenesis inhibition, and indicate a novel therapeutic target for type 2 diabetes mellitus.

Abstract 5415: Targeted depletion of polo-like kinase 1 alters metabolic regulation in melanoma

Abstract Polo-like kinase 1 (PLK1), a critical regulator of mitosis, is shown to be overexpressed in a variety of cancers. PLK1 overexpression has also been linked to poor disease prognosis and a lessened survival in cancer patients. Previously, we have shown that PLK1 is overexpressed in human melanoma and its inhibition causes significant anti-proliferative response in vitro (in multiple melanoma cells) as well as in vivo (in human melanoma xenografts). Further, in another study we demonstrated, based on a large-scale label-free comparative proteomics analysis, that PLK1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in human melanoma cells resulted in an alteration of certain metabolism-associated proteins with an associated decrease in cellular metabolism. In this study, to further explore the association between PLK1 and cellular metabolism, we utilized a doxycycline-inducible PLK1 knockdown approach in A375 melanoma cells coupled with a Human Glucose Metabolism PCR array that covers 84 key genes involved in the regulation and enzymatic pathways of glucose and glycogen metabolism. We found that PLK1 knockdown resulted in a significant downregulation of 29 genes and upregulation of 3 genes (more than 2 fold change) associated with cellular metabolism. IDH1, PDP2 and PCK1 were >3-fold downregulated while FBP1 was >7-fold upregulated. Through Ingenuity Pathway Analysis (IPA), we identified glycolysis and the pentose phosphate pathway as major canonical pathways altered by PLK1 inhibition. Further, IPA identified that PLK1 inhibition-modulated genes were largely associated with the proliferation of cells, where FBP1 appeared as a key regulatory player. PCK1 was found to be highly downregulated in our array, and IPA identified it as part of a monosaccharide regulation network. We further validated our data in vivo and found that BI 6727 treatment resulted in a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts. In addition, we observed a strong inverse correlation between PLK1 and FBP1 in multiple melanoma cell lines, with FBP1 expression significantly downregulated in a panel of melanoma cells compared to normal melanocytes. Moreover, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs 294T and G361 melanoma cells. Interestingly, in recent studies, FBP1 (fructose-bisphosphatase) that is a rate-limiting enzyme in gluconeogenesis, has been shown to function as a tumor suppressor in certain cancers. Overall, our study support the hypothesis that PLK1 is a regulator of metabolism maintenance that affects the melanoma cell growth. Citation Format: Rosie E. Gutteridge, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. Targeted depletion of polo-like kinase 1 alters metabolic regulation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5415. doi:10.1158/1538-7445.AM2017-5415

Abstract 4411: Loss of fructose-1,6-bisphosphatase expression induces altering glucose metabolism and tumor progression in hepatocellular carcinoma

Abstract Background: A recent study reported the loss of gluconeogenic capacity in hepatocellular carcinoma (HCC). Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in some cancers. However, the role of FBP1 in altered glucose metabolism in HCC was unclear. Therefore, the objective of this study was to examine the function and clinical significance of FBP1 expression in HCC. Materials and Methods: First, three independent cohorts totaling 594 cases of HCC (118 real-time RT-PCR data from our institution, 242 expression array data from GSE14520, and 234 RNA-sequencing data from The Cancer Genome Atlas (TCGA)) were analyzed to address clinical significance. Data from methylation arrays, SNP arrays, and whole-exome sequencing were also analyzed to investigate the regulation of FBP1 expression in the TCGA cohort. Second, we analyzed mRNA expression, promoter methylation, and DNA copy number profiles of 967 human cancer cell lines, including 27 liver cancer, in the Cancer Cell Line Encyclopedia. Third, we established HCC cell lines stably expressing FBP1 or empty vector control. We performed sphere formation assay and xenograft studies to evaluate the role of FBP1 on HCC progression. Furthermore, in order to assess the effect of FBP1 on altered glucose metabolism, isotopomer distribution analysis was performed using [U-13C] glucose. Finally, to validate the effects of FBP1 expression on survival, risk of recurrence, and glucose metabolism, we performed gene set enrichment analysis (GSEA). Results: Lower FBP1 expression associated with advanced tumor stage, poor overall survival (OS), and poor recurrence-free survival (RFS) in three independent HCC cohorts. For either OS or RFS in each cohort, this prognostic impact persisted, even after adjusting for tumor stage. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by GSEA. Conclusion: Our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Citation Format: Hiroaki Wakiyama, Hidenari Hirata, Keishi Sugimachi, Takaaki Masuda, Naoki Hayashi, Yohsuke Kuroda, Shuhei Ito, Hidetoshi Eguchi, Kotaro Terashima, Katsumi Sakamoto, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Loss of fructose-1,6-bisphosphatase expression induces altering glucose metabolism and tumor progression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4411. doi:10.1158/1538-7445.AM2017-4411

CREBH Maintains Circadian Glucose Homeostasis by Regulating Hepatic Glycogenolysis and Gluconeogenesis.

Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.

O-GlcNAcylation mediates the control of cytosolic phosphoenolpyruvate carboxykinase activity via Pgc1α.

PGC1α is a coactivator of many transcription factors and cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a key enzyme for gluconeogenesis. PGC1α interacts with the transcription factor PPARγ to stimulate PCK1 expression and thus de novo glucose synthesis. These proteins are not only important for central energy metabolism but also for supplying intermediates for other metabolic pathways, including lipidogenesis and protein synthesis and might therefore be important factors in the ethiopathogenesis of metabolic disorders like diabetes but also in other pathologies like cancer. Since polymorphisms in these proteins have been related to some phenotypic traits in animals like pigs and PGC1α G482S polymorphism increases fat deposition in humans, we have investigated the molecular basis of such effects focusing on a commonly studied polymorphism in pig Pgc1α, which changes a cysteine at position 430 (WT) of the protein to a serine (C430S). Biochemical analyses show that Pgc1α WT stimulates higher expression of human PCK1 in HEK293T and HepG2 cells. Paradoxically, Pgc1α WT is less stable than Pgc1α p.C430S in HEK293T cells. However, the study of different post-translational modifications shows a higher O-GlcNAcylation level of Pgc1α p.C430S. This higher O-GlcNAcylation level significantly decreases the interaction between Pgc1α and PPARγ demonstrating the importance of post-translational glycosylation of PGC1α in the regulation of PCK1 activity. This, furthermore, could explain at least in part the observed epistatic effects between PGC1α and PCK1 in pigs.

Invalidation of mitophagy by FBP1-mediated repression promotes apoptosis in breast cancer.

Fructose-1,6-bisphosphatase 1, a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor. However, the functions of fructose-1,6-bisphosphatase 1 in the regulation of mitophagy and apoptosis remain unknown. Here, we investigated the effects of fructose-1,6-bisphosphatase 1 on mitophagy and apoptosis as well as their underlying mechanisms in breast cancer cells. In this work, the messenger RNA and protein expression of various molecules were determined by quantitative realtime polymerase chain reaction and western blot, respectively. Gene-expression correlations were obtained from The Cancer Genome Atlas Breast Cancer database and analyzed using cBioPortal. The levels of cellular reactive oxygen species and apoptotic index were detected by flow cytometry. The mitochondrial membrane potentials were assessed with a JC-1 fluorescent sensor. Subcellular structures were observed under a transmission electron microscope. The intracellular distribution of translocase of outer membrane 20 was detected by immunofluorescence staining. Protein-protein interactions were analyzed by immunoprecipitation. Our results indicated that fructose-1,6-bisphosphatase 1 expression was negatively correlated with autophagy level in breast cancer. Fructose-1,6-bisphosphatase 1 restrained autophagy activity by increasing the level of p62 and decreasing the levels of LC3 and Beclin 1. Additionally, fructose-1,6-bisphosphatase 1 promoted cell apoptosis by upregulating the levels of intracellular ROS and expression of pro-apoptotic proteins such as cleaved PARP, cleaved Caspase 3, and Bax and downregulating the levels of anti-apoptotic proteins such as PARP, Caspase 3, and Bcl-2. Finally, fructose-1,6-bisphosphatase 1 limited the efficient removal of diseased mitochondria and reduced the messenger RNA and protein expressions of HIF-1α, BNIP3L/NIX, and BNIP3. More importantly, fructose-1,6-bisphosphatase 1 facilitated co-action between Bcl-2 and Beclin 1, which may be important in the mechanism of fructose-1,6-bisphosphatase 1-mediated mitophagy inhibition. In summary, loss of mitophagy by fructose-1,6-bisphosphatase 1-mediated repression promotes apoptosis in breast cancer.

Identification of a multienzyme complex for glucose metabolism in living cells

Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.

Aqueous root extracts of Dicoma anomala (Sond.) extenuates postprandial hyperglycaemia in vitro and its modulation on the activities of carbohydrate-metabolizing enzymes in streptozotocin-induced diabetic Wistar rats

Dicoma anomala Sond (Asteraceae) is widely incorporated as a treatment for diabetes mellitus among the Basotho tribe of the Eastern Free State Province, South Africa. The study examined the antidiabetic effect of the plant via the in vitro inhibition of α-amylase, and α-glucosidase as well as against streptozotocin (STZ)-induced diabetic Wistar rats. The in vitro antidiabetic activity of the plant is via the inhibition of α-amylase, and α-glucosidase using water, ethanol, hydro-ethanol and methanol extracts. The effect of administration of aqueous root extract of Dicoma anomala (AQRED) at 125, 250, and 500mg/kg bodyweight (b.w.) on water consumption, feed intake, body-weight, blood glucose, carbohydrate-metabolizing enzymes, antioxidant enzymes, glycosylated haemoglobin and lipid profiles was determined in STZ (60mg/kg b.w.)-induced diabetic rats with comparison with glibenclamide (5mg/kg b.w.). While all the extracts of D. anomala showed activity against α-amylase and α-glucosidase, water extract revealed the most effective inhibition with an IC50 of 51.90 and 27.41μg/mL respectively. The water extract displayed competitive and non-competitive inhibitors of α-amylase and α-glucosidase respectively. AQRED reversed towards normal control the elevated food/water intake, blood glucose levels, lipid peroxidation, lipid profiles, glycosylated haemoglobin and activities of gluconeogenesis enzymes with a concomitant decrease in body-weight, activities of enzymatic antioxidants, glycolytic enzymes as well as the high-density lipoprotein-cholesterol level brought-about by STZ administration. The result of our findings proved the antihyperglycaemic activity of the plant and therefore validates the folkloric usage of the herb.

Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex.

Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.

Pro(moting) the Turnover of Gluconeogenic Enzymes by a New Branch of the N-end Rule Pathway

The N-end rule pathway is a set of protein degradation systems that link the in vivo stability of a protein to its N-terminal residue. A recent paper from Alexander Varshavsky's laboratory [1] identifies a new branch of the N-end rule pathway that specifically recognizes the N-terminal Pro residue of key gluconeogenesis enzymes.

Inhibition of liver alanine aminotransferase and aspartate aminotransferase by hesperidin and its aglycone hesperetin: An in vitro and in silico study

AimsThis study aimed to investigate the inhibitory effects of two natural flavonoids, hesperetin (HT) and hesperidin (HD), on two gluconeogenesis enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and their possible mechanisms of action. Main methodsRat liver incubated with different concentrations of HT and HD was used to measure enzyme activities spectrophotometrically, based on monitoring the oxidation of NADH to NAD+ at 340nm. Molecular docking simulation was also applied to reveal the molecular mechanism of the inhibition caused by HT and HD. Key findingsBoth flavonoids demonstrated inhibitory effects against the enzyme activities, with IC50 values of 153.9 and 68.88μM for HT-ALT and HD-ALT treatment respectively. Likewise, the IC50 values of 85.29μM for HT-AST and 110.3μM for HD-AST were obtained from spectrophotometric results. ConclusionThe docking simulation revealed that HT and HD block the enzyme entrance channel and prevent the substrates from accessing the enzyme active sites. Having prevented production of pyruvate, α-ketoglutarate, and the oxaloacetate, these two compounds inhibit hepatic gluconeogenesis and consequently, hinder the progression of diabetes. SignificanceThis study suggests that HT and HD may be considered as leading compounds for designing safe and effective drugs in management of increased ALT and AST-related disorders specially diabetes.

MAGE-TRIM28 complex promotes the Warburg effect and hepatocellular carcinoma progression by targeting FBP1 for degradation

Hepatocellular carcinoma (HCC) is one of the leading cause of cancer death in the world. Fructose-1,6-biphosphatase (FBP1), a rate-limiting enzyme in gluconeogenesis, has been identified recently as a tumor suppressor in HCC and other cancer types. In this study, we demonstrated that the tripartite motif-containing protein 28 (TRIM28) binds directly to and promotes FBP1 for ubiquitination and degradation. MAGE-A3 and MAGE-C2, which are known to be overexpressed in HCC, can enhance TRIM28-dependent degradation of FBP1 by forming ubiquitin ligase complexes with TRIM28. We further showed that expression of TRIM28 increased glucose consumption and lactate production by promoting FBP1 degradation in HCC cells and that FBP1 is a key mediator of TRIM28-induced HCC growth in culture and in mice. Moreover, we demonstrated that FBP1 and TRIM28 protein levels inversely correlated in HCC patient specimens. Finally, we showed that the proteasome inhibitor bortezomib mitigated the Warburg effect by inhibiting FBP1 degradation in HCC. Collectively, our findings not only identify oncogenic MAGE-TRIM28 complex-mediated proteasome degradation of FBP1 as a key mechanism underlying downregulation of FBP1 proteins in HCC, but also reveal that MAGE-TRIM28-regulated reprogramming of cancer cell metabolism and HCC tumorigenesis is mediated, at least in part, through FBP1 degradation.

Insulin-induced inhibition of gluconeogenesis genes, including glutamic pyruvic transaminase 2, is associated with reduced histone acetylation in a human liver cell line

ObjectivesHepatic glutamic pyruvic transaminase (GPT; also known as alanine aminotransferase) is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. It is also used as a blood biomarker for hepatic damage. In this study, we investigated whether insulin regulates GPT expression, as it does for other gluconeogenesis genes, and if this involves the epigenetic modification of histone acetylation. MethodsHuman liver-derived HepG2 cells were cultured with 0.5–100nM insulin for 8h, and the mRNA expression of GPT, glutamic–oxaloacetic transaminase (GOT), γ-glutamyltransferase (GGT), PCK1, G6PC and FBP1 was measured. We also investigated the extent of histone acetylation around these genes. ResultsInsulin suppressed the mRNA expression of gluconeogenesis genes (GPT2, GOT1, GOT2, GGT1, GGT2, G6PC, and PCK1) in HepG2 cells in a dose-dependent manner. mRNA levels of GPT2, but not GPT1, were decreased by insulin. Histone acetylation was also reduced around GPT2, G6PC, and PCK1 in response to insulin. ConclusionThe expression of GPT2 and other gluconeogenesis genes such as G6PC and PCK1 was suppressed by insulin, in association with decreases in histone H3 and H4 acetylation surrounding these genes.

Inhibiting histone deacetylases suppresses glucose metabolism and hepatocellular carcinoma growth by restoring FBP1 expression

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers in the world. Elevated glucose metabolism in the availability of oxygen, a phenomenon called the Warburg effect, is important for cancer cell growth. Fructose-1,6-bisphosphatase (FBP1) is a rate-limiting enzyme in gluconeogenesis and is frequently lost in various types of cancer. Here, we demonstrated that expression of FBP1 was downregulated in HCC patient specimens and decreased expression of FBP1 associated with poor prognosis. Low expression of FBP1 correlated with high levels of histone deacetylase 1 (HDAC1) and HDAC2 proteins in HCC patient tissues. Treatment of HCC cells with HDAC inhibitors or knockdown of HDAC1 and/or HDAC2 restored FBP1 expression and inhibited HCC cell growth. HDAC-mediated suppression of FBP1 expression correlated with decreased histone H3 lysine 27 acetylation (H3K27Ac) in the FBP1 enhancer. Restored expression of FBP1 decreased glucose reduction and lactate secretion and inhibited HCC cell growth in vitro and tumor growth in mice. Our data reveal that loss of FBP1 due to histone deacetylation associates with poor prognosis of HCC and restored FBP1 expression by HDAC inhibitors suppresses HCC growth. Our findings suggest that repression of FBP1 by HDACs has important implications for HCC prognosis and treatment.

L-Glutamine supplementation promotes an improved energetic balance in Walker-256 tumor-bearing rats.

We evaluated the effects of supplementation with oral l-glutamine in Walker-256 tumor-bearing rats. A total of 32 male Wistar rats aged 54 days were randomly divided into four groups: rats without Walker-256 tumor, that is, control rats (C group); control rats supplemented with l-glutamine (CG group); Walker-256 tumor rats without l-glutamine supplementation (WT group); and WT rats supplemented with l-glutamine (WTG group). l-Glutamine was incorporated into standard food at a proportion of 2 g/100 g (2%). After 10 days of the experimental period, the jejunum and duodenum were removed and processed. Protein expression levels of key enzymes of gluconeogenesis, that is, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, were analyzed by western blot and immunohistochemical techniques. In addition, plasma corticosterone, glucose, insulin, and urea levels were evaluated. The WTG group showed significantly increased plasma glucose and insulin levels ( p < 0.05); however, plasma corticosterone and urea remained unchanged. Moreover, the WTG group showed increased immunoreactive staining for jejunal phosphoenolpyruvate carboxykinase and increased expression of duodenal glucose-6-phosphatase. Furthermore, the WTG group presented with less intense cancer cachexia and slower tumor growth. These results could be attributed, at least partly, to increased intestinal gluconeogenesis and insulinemia, and better glycemia maintenance during fasting in Walker-256 tumor rats on a diet supplemented with l-glutamine.

ATRA modulates gluconeogenesis and triglyceride biosynthesis pathway of liver in elastase induced emphysematous rats

Emphysema is a chronic pulmonary disorder characterized by the destruction and enlargement of the gas exchanging units known as alveoli. Chronic inflammation is one of the critical features associated with pathogenesis of emphysema. The inflammatory mediators generated during emphysema are thought to be infiltrated into the circulatory system, leading to systemic inflammation and thus, affecting other vital organs. The potential role of ATRA, an anti-inflammatory bio-molecule, towards molecular mechanisms governing systemic inflammation due to inflammatory mediator’s overspills in emphysema is poorly understood. Therefore, the present study deals with investigating the potential effect of ATRA on physiological functions of liver tissue in an established elastase induced emphysema rat model. Three experimental groups (i.e., control, SS; emphysema, ES; and therapy, EA) were prepared. Subsequently, liver from each rat was collected for elastase activity assay, histopathology, real-time PCR based mRNA expression analyses, total lipid estimation and western blot analysis. ATRA supplementation in emphysema group decreases elastase activity. Liver histopathology photomicrographs clearly shows an increase in number of Kupffer cells in ES than SS, while, a decrease in number of Kupffer cells was found in EA. Furthermore, upon validation at mRNA and protein level, expressions of TNF-α, enzymes of gluconeogenesis and triglyceride biosynthesis pathway were significantly increased in ES while decreased in EA when compared to SS. Therapeutic supplementation of ATRA posed a beneficial effect in maintaining the normal architecture of liver tissue along with modulation of gluconeogenesis and triglyceride biosynthesis pathways.

Sirtinol promotes PEPCK1 degradation and inhibits gluconeogenesis by inhibiting deacetylase SIRT2

Phosphoenolpyruvate carboxykinase 1 (PEPCK1) is the critical enzyme for gluconeogenesis and is linked with type II diabetes. Previous studies have found that SIRT2, a deacetylase, plays an important role in deacetylating PEPCK1 and little is known about the anti-diabetic activity of SIRT2 inhibitors. In this study, we investigated the anti-diabetic effects of sirtinol, a SIRT2 inhibitor, on cell gluconeogenesis in vivo and in vitro. Immunoblotting analysis revealed that sirtinol significantly decreased the protein level of PEPCK1, and was accompanied by the hyperacetylation of PEPCK1 as well as decreased glucose output in a dose-dependent manner. Furthermore, sirtinol exerted little impact on endogenous PEPCK1 levels in SIRT2-knockdown cells. The in vitro experiments further confirmed the in vivo data; sirtinol decreased liver PEPCK1 protein level and prevented pyruvate-induced blood glucose from increasing. Based on our results, the rate-limiting enzyme PEPCK1 is the primary target of sirtinol, and the inhibition of SIRT2 activity may play an important role in cell gluconeogenesis. Thus, SIRT2 may be a novel molecular target for diabetes therapy and may thus shed light on the underlying diabetes treatment mechanisms of sirtinol.

Nur77 suppresses hepatocellular carcinoma via switching glucose metabolism toward gluconeogenesis through attenuating phosphoenolpyruvate carboxykinase sumoylation

Gluconeogenesis, an essential metabolic process for hepatocytes, is downregulated in hepatocellular carcinoma (HCC). Here we show that the nuclear receptor Nur77 is a tumour suppressor for HCC that regulates gluconeogenesis. Low Nur77 expression in clinical HCC samples correlates with poor prognosis, and a Nur77 deficiency in mice promotes HCC development. Nur77 interacts with phosphoenolpyruvate carboxykinase (PEPCK1), the rate-limiting enzyme in gluconeogenesis, to increase gluconeogenesis and suppress glycolysis, resulting in ATP depletion and cell growth arrest. However, PEPCK1 becomes labile after sumoylation and is degraded via ubiquitination, which is augmented by the p300 acetylation of ubiquitin-conjugating enzyme 9 (Ubc9). Although Nur77 attenuates sumoylation and stabilizes PEPCK1 via impairing p300 activity and preventing the Ubc9-PEPCK1 interaction, Nur77 is silenced in HCC samples due to Snail-mediated DNA methylation of the Nur77 promoter. Our study reveals a unique mechanism to suppress HCC by switching from glycolysis to gluconeogenesis through Nur77 antagonism of PEPCK1 degradation.