A Pck1-directed glycogen metabolic program regulates formation and maintenance of memory CD8+ T cells.

CD8+ memory T (Tm) cells are fundamental for protective immunity against infections and cancers 1-5 . Metabolic activities are crucial in controlling memory T-cell homeostasis, but mechanisms linking metabolic signals to memory formation and survival remain elusive. Here we show that CD8+ Tm cells markedly upregulate cytosolic phosphoenolpyruvate carboxykinase (Pck1), the hub molecule regulating glycolysis, tricarboxylic acid cycle and gluconeogenesis, to increase glycogenesis via gluconeogenesis. The resultant glycogen is then channelled to glycogenolysis to generate glucose-6-phosphate and the subsequent pentose phosphate pathway (PPP) that generates abundant NADPH, ensuring high levels of reduced glutathione in Tm cells. Abrogation of Pck1-glycogen-PPP decreases GSH/GSSG ratios and increases levels of reactive oxygen species (ROS), leading to impairment of CD8+ Tm formation and maintenance. Importantly, this metabolic regulatory mechanism could be readily translated into more efficient T-cell immunotherapy in mouse tumour models.

Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation.

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the beta-cells of the islets.

The phosphoenolpyruvate carboxykinase of Mycobacterium tuberculosis induces strong cell-mediated immune responses in mice.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes guanosine or adenosine mononucleotide-dependent reversible conversion of oxaloacetate (OAA) and phosphoenolpyruvate (PEP). Mycobacterium (M) tuberculosis possesses a putative GTP-dependent PEPCK. To analyze the immune responses caused by PEPCK, the effects of PEPCK on the induction of CD4(+) T cells and cytokines such as IFN-gamma, IL-12 and TNF-alpha were evaluated in mice. It was found that the number of CD4(+) T cells was increased in the PEPCK immunized mice although the change of the number of CD8(+) T cells was not significant. The cytokines IFN-gamma, IL-12 and TNF-alpha were increased significantly in the mice immunized with PEPCK than those of incomplete adjuvant. These characteristics were further demonstrated in the mice infected by pckA mutated BCG strain. The results indicate that PEPCK can effectively induce cell-mediated immune response by increasing activity of cytokines and PEPCK may be a promising new subunit vaccine candidate for tuberculosis.

Adiponectin and its receptors in patients with chronic hepatitis C.

BACKGROUND/AIMS: There is increasing interest in the influence of excess body weight and associated metabolic factors on the liver. In patients with non-alcoholic steatohepatitis, lower levels of adiponectin were associated with higher grades of hepatic steatosis and necroinflammatory activity, suggesting a pathophysiological role for this adipokine in liver disease. METHODS: We studied 194 consecutive patients with untreated chronic HCV, to assess the relationship between adiponectin and its receptors and hepatic steatosis, fibrosis and inflammation. RESULTS: Significant negative correlations between serum adiponectin and male gender, body mass index and serum insulin were observed. However, there was no association between serum adiponectin and stage of fibrosis and lower levels of serum adiponectin were associated with the presence of steatosis in males only. In contrast, there was a significant increase in serum adiponectin and hepatic adiponectin immunoreactivity with increasing inflammation. The hepatic mRNA expression of the adiponectin receptors, AdipoR1 and AdipoR2, displayed significant but opposite associations with phosphoenolpyruvate carboxykinase (PEPCK) gene expression, a substitute marker of hepatic insulin sensitivity. CONCLUSIONS: In patients with chronic HCV, adiponectin was associated with steatosis only in males and was paradoxically increased with inflammation. Our results suggest that the role of adiponectin in chronic liver diseases may be linked to gender and etiology.

Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope.

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.

Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides.

A rabbit antiserum was raised against phosphoenolpyruvate carboxykinase (PCK) purified from Urochloa panicoides, a PCK-type C4 monocot. The antiserum was used to screen a cDNA expression library constructed from U. panicoides leaf poly(A)+RNA. Inserts from immunoreactive clones were used to rescreen the library and obtain three overlapping cDNAs comprising a 2220 bp composite sequence. The single complete open reading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68,474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainly due to an N-terminal extension of greater than 65 residues, and reveals high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and divalent cation binding. The anti-PCK antiserum recognises a 69 kDa polypeptide on immunoblots of either purified PCK or U. panicoides leaf extracts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunoreactive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides arise from the breakdown of PCK during isolation. Northern blot analysis indicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradually during greening, reaching maximum levels after about 84 h.

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression.

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.

Enhancement of antigenic site detection with gold labeled secondary and tertiary antibodies using the immunogold-silver staining method.

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 microns frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver.

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35-1.0 micron) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 microns. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae.

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.

Regulation of phosphoenolpyruvate carboxykinase and fatty acid synthetase in brown fat of suckling rats.

Infant rats were injected with prednisolone (0.5-5 mg/100 g wt). This caused phosphoenolpyruvate carboxykinase (PEPCK) activity to rise in liver and to decrease in brown fat. Fatty acid synthetase (FAS) activity remained unchanged in liver but increased in brown fat. A single injection of prednisolone caused hepatic PEPCK activity to remain elevated for at least 7 days. Brown fat FAS also remained high for that period. However, brown fat PEPK activity returned to normal on the third day after the injection. A single injection of prednisolone or cortisone to 5-day-old rats caused a transient elevation of the blood level of insulin and a prolonged decrease in that of growth hormone. No effect on the level of glucagon was noted. Injections of insulin had effects similar to those of prednisolone, i.e. a rise in hepatic and a fall in brown fat PEPCK. Using antibodies prepared to hepatic PEPCK it was shown that the observed changes were due to changes in the rate of synthesis of the enzyme. Using actinomycin D indirect evidence was obtained that changes in FAS activity of brown fat were also due to changes in the synthetic rate.

The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro.

1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5--7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.

Effects of cyclic adenosine monophosphate, dexamethasone and insulin on phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 hepatoma cells.

Antiserum prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is shown to specifically precipitate the enzyme from Reuber H-35 cells. Synthesis of phosphoenolpyruvate carboxykinase, as measured immunochemically, is increased by dibutyryl cAMP and dexamethasone, the nucleotide maximally producing a sixfold and the glucocorticoid a threefold change in rate. Studies with actinomycin D, cordycepin, and cycloheximide suggest dibutyryl cAMP acts at a translational or post-transcriptional site. Insulin prevents the increase in synthesis of phosphoenolpyruvate carboxykinase produced by either dibutyryl cAMP or dexamethasone. This antagonism is concentration dependent and does not require the simultaneous presence of glucose, pointing to a direct effect of the hormone on liver enzyme induction. It is suggested that hepatic phosphoenolpyruvate carboxykinase activity is regulated predominantly by the antagonistic interaction of cAMP (glucagon) and insulin on enzyme synthesis.