Distribution and prognostic significance of gluconeogenesis and glycolysis in lung cancer.

Inhibition of glycolysis has been considered as a therapeutic approach in aggressive cancers including lung cancer. Abbreviated gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (PEPCK), was recently discovered to partially circumvent the need for glycolysis in lung cancer cells. However, the interplay of glycolysis and gluconeogenesis in lung cancer is still poorly understood. Here, we analyzed the expression of GLUT1, the prime glucose transporter, and of PCK1 and PCK2, the cytoplasmic and mitochondrial isoforms of PEPCK, in 450 samples of non-small cell lung cancer (NSCLC) and in 54 NSCLC metastases using tissue microarrays and whole tumor sections. Spatial distribution was assessed by automated image analysis. Additionally, glycolytic and gluconeogenic gene expression was inferred from The Cancer Genome Atlas (TCGA) datasets. We found that PCK2 was preferentially expressed in the lung adenocarcinoma subtype, while GLUT1 expression was higher in squamous cell carcinoma. GLUT1 and PCK2 were inversely correlated, GLUT1 showing elevated expression in larger tumors while PCK2 was highest in smaller tumors. However, a mixed phenotype showing the presence of both, glycolytic and gluconeogenic cancer cells was frequent. In lung adenocarcinoma, PCK2 expression was associated with significantly improved overall survival, while the opposite was found for GLUT1. The metabolic tumor microenvironment and the 3-dimensional context play an important role in modulating both pathways, since PCK2 expression preferentially occurred at the tumor margin and hypoxia regulated both, glycolysis and gluconeogenesis, in NSCLC cells in vitro, albeit in opposite directions. PCK1/2 expression was enhanced in metastases compared to primary tumors, possibly related to the different environment. The results of this study show that glycolysis and gluconeogenesis are activated in NSCLC in a tumor size and oxygenation modulated manner and differentially correlate with outcome. The frequent co-activation of gluconeogenesis and glycolysis in NSCLC should be considered in potential future therapeutic strategies targeting cancer cell metabolism.

Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase-1 expression in fetal baboon hepatocytes in vitro.

BACKGROUND: The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared with ad libitum-fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK-1 expression. METHODS: Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively. RESULTS: Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. CONCLUSIONS: Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.

Effects of non-esterified fatty acids on the gluconeogenesis in bovine hepatocytes.

Dairy cows experience an increased demand for glucose to support milk production. However, negative energy balance is a common condition in peripartum cows. In response, fat mobilization provides non-esterified fatty acids (NEFAs) for oxidation in the liver to generate ATP. To investigate the effects of NEFAs on gluconeogenesis, the expression and enzyme activity of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPCK) in cultured bovine hepatocytes were evaluated by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that PC and PEPCK mRNA levels were marked decreased when the NEFAs concentrations exceeded 0.5 and 1.5 mmol/l, respectively. The PC and PEPCK enzyme activity showed significantly decreased when the NEFAs concentrations exceeded 1.5 and 0.5 mmol/l, respectively. These findings indicate that high circulating levels of NEFAs inhibit hepatocyte gluconeogenesis, thereby promoting negative energy balance.

Spontaneous activity, economy of activity, and resistance to diet-induced obesity in rats bred for high intrinsic aerobic capacity.

Though obesity is common, some people remain resistant to weight gain even in an obesogenic environment. The propensity to remain lean may be partly associated with high endurance capacity along with high spontaneous physical activity and the energy expenditure of activity, called non-exercise activity thermogenesis (NEAT). Previous studies have shown that high-capacity running rats (HCR) are lean compared to low-capacity runners (LCR), which are susceptible to cardiovascular disease and metabolic syndrome. Here, we examine the effect of diet on spontaneous activity and NEAT, as well as potential mechanisms underlying these traits, in rats selectively bred for high or low intrinsic aerobic endurance capacity. Compared to LCR, HCR were resistant to the sizeable increases in body mass and fat mass induced by a high-fat diet; HCR also had lower levels of circulating leptin. HCR were consistently more active than LCR, and had lower fuel economy of activity, regardless of diet. Nonetheless, both HCR and LCR showed a similar decrease in daily activity levels after high-fat feeding, as well as decreases in hypothalamic orexin-A content. The HCR were more sensitive to the NEAT-activating effects of intra-paraventricular orexin-A compared to LCR, especially after high-fat feeding. Lastly, levels of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in the skeletal muscle of HCR were consistently higher than LCR, and the high-fat diet decreased skeletal muscle PEPCK-C in both groups of rats. Differences in muscle PEPCK were not secondary to the differing amount of activity. This suggests the possibility that intrinsic differences in physical activity levels may originate at the level of the skeletal muscle, which could alter brain responsiveness to neuropeptides and other factors that regulate spontaneous daily activity and NEAT.

Novel mechanism for plasma glucose-lowering action of metformin in streptozotocin-induced diabetic rats.

To better understand the insulin-independent plasma glucose-lowering action of metformin, we used streptozotocin (STZ)-induced diabetic rats to investigate the possible mechanisms. Oral intake of metformin decreased the plasma glucose of STZ-induced diabetic rats with a parallel increase of plasma beta-endorphin-like immunoreactivity (BER). Mediation of opioid mu-receptors in the action of metformin was identified by the blockade of receptors with antagonist in STZ-induced diabetic rats and the failure of action in opioid mu-receptor knockout diabetic mice. Release of BER from adrenal glands by metformin was characterized, using bilateral adrenalectomy and the release of BER from isolated adrenal medulla of STZ-induced diabetic rats. Repeated treatment with metformin in STZ-induced diabetic rats increased the mRNA and protein levels of GLUT-4 in soleus muscle that was blocked by naloxonazine. Reduction of the mRNA or protein levels of hepatic PEPCK was also impeded in the same group of STZ-induced diabetic rats. In conclusion, our results provide novel mechanisms for the plasma glucose-lowering action of metformin, via an increase of beta-endorphin secretion from adrenal glands to stimulate opioid mu-receptor linkage, leading to an increase of GLUT-4 gene expression and an attenuation of hepatic PEPCK gene expression in STZ-induced diabetic rats.

Merit of physical exercise to reverse the higher gene expression of hepatic phosphoenolpyruvate carboxykinase in obese Zucker rats.

Effects of endurance training on the phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme of gluconeogenesis, were studied in the obese Zucker rats. We used a moderate exercise program consisting of treadmill running at 20 m/min and 0-degree gradient for 1 h/day, 7 days/week, for 8 weeks. At the end of the experimental period, insulin action on glucose disposal rate was measured using the glucose-insulin index, the product of the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test. Furthermore, changes of hepatic PEPCK gene expression were detected using reverse transcriptase polymerase chain reaction to assay the mRNA level and Western blot analysis to detect the protein level. Different to sedentary obese rats, an elevation in the value of glucose-insulin index from the exercised obese rats declined, indicating the marked effect of regular moderate exercise on the improvement of insulin sensitivity in this insulin resistant animal model. Moreover, the diabetes-related elevation in mRNA level and protein content of hepatic PEPCK were observed in non-exercise obese groups but they were markedly reduced by exercise training. In addition, chronic exercise training enhanced the insulin sensitivity of lean Zucker rats, since the value of glucose-insulin index was lower than that of untrained lean groups. Also, the hepatic PEPCK gene expressions both the mRNA and protein levels were reduced in exercised lean Zucker rats as compared with their sedentary littermates. These results suggest that modulation of hepatic PEPCK gene expression by chronic exercise training might be related to the enhancement of insulin sensitivity. Thus, endurance exercise training could aid in the prevention and/or treatment of individuals with insulin resistance.

Non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling 3 (SOCS-3) in patients with chronic hepatitis C, viral genotype 1.

BACKGROUND: Interferon alpha (IFN-alpha) activated cellular signalling is negatively regulated by inhibitory factors, including the suppressor of cytokine signalling (SOCS) family. The effects of host factors such as obesity on hepatic expression of these inhibitory factors in subjects with chronic hepatitis C virus (HCV) are unknown. OBJECTIVES: To assess the independent effects of obesity, insulin resistance, and steatosis on response to IFN-alpha therapy and to determine hepatic expression of factors inhibiting IFN-alpha signalling in obese and non-obese subjects with chronic HCV. METHODS: A total of 145 subjects were analysed to determine host factors associated with non-response to antiviral therapy. Treatment comprised IFN-alpha or peginterferon alpha, either alone or in combination with ribavirin. In a separate cohort of 73 patients, real time-polymerase chain reaction was performed to analyse hepatic mRNA expression. Immunohistochemistry for SOCS-3 was performed on liver biopsy samples from 38 patients with viral genotype 1 who had received antiviral treatment. RESULTS: Non-response (NR) to treatment occurred in 55% of patients with HCV genotypes 1 or 4 and 22% with genotypes 2 or 3. Factors independently associated with NR were viral genotype 1/4 (p < 0.001), cirrhosis on pretreatment biopsy (p = 0.025), and body mass index > or = 30 kg/m2 (p = 0.010). Obese subjects with viral genotype 1 had increased hepatic mRNA expression of phosphoenolpyruvate carboxy kinase (p = 0.01) and SOCS-3 (p = 0.047), in comparison with lean subjects. Following multivariate analysis, SOCS-3 mRNA expression remained independently associated with obesity (p = 0.023). SOCS-3 immunoreactivity was significantly increased in obesity (p = 0.013) and in non-responders compared with responders (p = 0.014). CONCLUSIONS: In patients with chronic HCV viral genotype 1, increased expression of factors that inhibit interferon signalling may be one mechanism by which obesity reduces the biological response to IFN-alpha.

Dipeptidyl peptidase IV inhibitor treatment stimulates beta-cell survival and islet neogenesis in streptozotocin-induced diabetic rats.

Recent studies into the physiology of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have added stimulation of beta-cell growth, differentiation, and cell survival to well-documented, potent insulinotropic effects. Unfortunately, the therapeutic potential of these hormones is limited by their rapid enzymatic inactivation in vivo by dipeptidyl peptidase IV (DP IV). Inhibition of DP IV, so as to enhance circulating incretin levels, has proved effective in the treatment of type 2 diabetes both in humans and in animal models, stimulating improvements in glucose tolerance, insulin sensitivity, and beta-cell function. We hypothesized that enhancement of the cytoprotective and beta-cell regenerative effects of GIP and GLP-1 might extend the therapeutic potential of DP IV inhibitors to include type 1 diabetes. For testing this hypothesis, male Wistar rats, exposed to a single dose of streptozotocin (STZ; 50 mg/kg), were treated twice daily with the DP IV inhibitor P32/98 for 7 weeks. Relative to STZ-injected controls, P32/98-treated animals displayed increased weight gain (230%) and nutrient intake, decreased fed blood glucose ( approximately 26 vs. approximately 20 mmol/l, respectively), and a return of plasma insulin values toward normal (0.07 vs. 0.12 nmol/l, respectively). Marked improvements in oral glucose tolerance, suggesting enhanced insulin secretory capacity, were corroborated by pancreas perfusion and insulin content measurements that revealed two- to eightfold increases in both secretory function and insulin content after 7 weeks of treatment. Immunohistochemical analyses of pancreatic sections showed marked increases in the number of small islets (+35%) and total beta-cells (+120%) and in the islet beta-cell fraction (12% control vs. 24% treated) in the treated animals, suggesting that DP IV inhibitor treatment enhanced islet neogenesis, beta-cell survival, and insulin biosynthesis. In vitro studies using a beta-(INS-1) cell line showed a dose-dependent prevention of STZ-induced apoptotic cell-death by both GIP and GLP-1, supporting a role for the incretins in eliciting the in vivo results. These novel findings provide evidence to support the potential utility of DP IV inhibitors in the treatment of type 1 and possibly late-stage type 2 diabetes.

Metformin-like effects of Quei Fu Di Huang Wan, a Chinese herbal mixture, on streptozotocin-induced diabetic rat.

Effect on plasma glucose concentration of Quei Fu Di Huang Wan (Quei Fu DHW), the herbal mixture widely used to treat diabetic disorder in Chinese traditional medicine, was investigated in diabetic rats deficient in insulin. Changes of plasma glucose in streptozotocin-induced diabetic rats (STZ-diabetic rats) receiving repeated oral administration of Quei Fu DHW were determined. Also, the mRNA level (by Northern blotting) and protein level (by Western blotting) of phosphoenolpyruvate carboxykinase (PEPCK) in liver from STZ-diabetic rats were measured to compare differences between groups receiving repeated oral administration of Quei Fu DHW, metformin, and two active herbs (Zou Guei or Fuzei) at effective dosages. In STZ-diabetic rats, acute oral administration of Quei Fu DHW decreased the plasma glucose level significantly in a dose-dependent manner from 5 mg/kg to 26.0 mg/kg. Similar treatment with Quei Fu DHW also brought on a plasma glucose-lowering effect in normal rats, although the effectiveness was not as significant as in STZ-diabetic rats. Repeated oral treatment of Quei Fu DHW at 26 mg/kg every 8 h, three times daily for 3 days, produced a plasma glucose-lowering activity similar to that of metformin-treatment in STZ-diabetic rats. Oral administration of Zou Guei (Cinnamomi Cortex) or Fuzei (Aconiti Tuber), the individual constituent of Quei Fu DHW, at the dose of 50 mg/kg into STZ-diabetic rats for 3 days normalized hyperglycemia. Similar to the repeated treatment with Quei Fu DHW, Fuzei at the effective dose reversed the elevated mRNA and protein levels of PEPCK in liver from STZ-diabetic rats. This is consistent with findings that metformin restored the increased gene expression of PEPCK in liver from STZ-diabetic rats. However, the gene expression of PEPCK in STZ-diabetic rats was not influenced by similar treatment with Zou Guei. The present study found that oral administration of Quei Fu DHW could decrease hepatic gluconeogenesis in a way similar to metformin in lowering plasma glucose in diabetic rats lacking insulin. Thus, this preparation may be a helpful adjuvant for the treatment of diabetic disorders in clinical practice.

The relationship between differentiation and proliferation in late gestation fetal rat hepatocytes.

Hepatocyte proliferation and differentiation occur simultaneously during late mammalian gestation. We hypothesized that regulation of hepatocyte growth and differentiation would be coordinated in late gestation fetal hepatocyte cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for alpha-fetoprotein (AFP), three markers of enzymic differentiation (glucokinase [GK], phosphoenolpyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), and a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was assessed using immunocytochemical detection of proliferating cell nuclear antigen (PCNA) or 5-bromo-2'-deoxy-uridine (BrdU) incorporation into DNA. Fetal hepatocyte cultures consisted of a heterogeneous population of cells, slightly more than half of which were proliferative under defined, growth factor-free conditions. These cultures were heterogeneous for AFP expression. There was no correlation between the expression of AFP and PCNA or AFP and S-phase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CAM. In addition, the differentiation status of cultured fetal hepatocytes was unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor alpha (TGFalpha), and hepatocyte growth factor (HGF). Finally, absence of any correlation between proliferation and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results indicate that the developmental program governing differentiation of late gestation fetal rat hepatocytes is independent from mechanisms controlling proliferation.

Expression and subcellular distribution of phosphoenolpyruvate carboxykinase in primary cultures of rabbit kidney proximal tubule cells: comparative study with renal and hepatic PEPCK in vivo.

The behaviour of the phosphoenolpyruvate carboxykinase (PEPCK) in rabbit proximal tubule cells in primary culture was investigated and compared with renal and hepatic PEPCK in vivo. The enzyme activity decreased rapidly in rabbit proximal tubule cells developed in hormonally defined medium supplemented with glucose and insulin. In this condition, the cytosolic form disappears with time. Without glucose and insulin, the subcellular location of PEPCK is similar to the location observed in proximal tubule freshly isolated and in renal cortex, with approx. 50% of mitochondrial form and approx. 50% of cytosolic form. However, the levels of mRNA that encode the cytosolic PEPCK are not detectable in cell cultures, whatever the medium composition. Treatment with dibutyryl cAMP caused a 14-fold induction of PEPCK mRNA in 6 h. This result indicates that the transcription of cytosolic PEPCK can be induced in cell cultures. Lactate or pyruvate additions did not modify the levels of PEPCK mRNA whereas specific activity increased rapidly, suggesting an activation of an inactive form in cell cultures. Moreover, lactate induced increased specific activity of the sole mitochondrial form while pyruvate induced increased specific activities of both mitochondrial and cytosolic form. Thus, subcellular location of PEPCK in rabbit proximal tubule cells appears to be modulated by the available substrate in culture medium. This observation parallels the changes observed in vivo since a modification of subcellular location of this enzyme was seen between fed and fasted rabbit, when subcellular distribution remains similar between fed and starved rats. Moreover, in the fasted liver of rabbit, a decrease of the mitochondrial PEPCK specific activity is seen concomitant with an increase in cytosolic PEPCK activity. These results point out the relative contributions of the cytosolic and mitochondrial PEPCK to rabbit gluconeogenesis.

Decreased gluconeogenesis and increased glucose disposal without hyperinsulinemia in 10-day-old rats with endotoxic shock.

Glucose dyshomeostasis is a common and life-threatening sign of endotoxic shock in the newborn. In this study, liver gluconeogenesis was evaluated in 10-day-old rats with endotoxic shock using the isolated perfused liver. Phosphoenolpyruvate carboxykinase (PEPCK) activity and PEPCK mRNA abundance were measured to confirm altered gluconeogenesis. Glucose disposal was also evaluated by a glucose tolerance test. Twenty-four-hour-fasted rats were studied to enhance gluconeogenesis and decrease glucose disposal. Rats received an intraperitoneal (IP) injection as follows: group 1 (fed-saline), 0.2 mL saline in fed rats; group 2 (fed-LPS), 0.1 mg/kg Salmonella enteritidis lipopolysaccharide (LPS) in fed rats; group 3 (fasted-saline), 0.2 mL saline in fasted rats; and group 4 (fasted-LPS), 0.1 mg/kg LPS in fasted rats. Isolated liver perfusion, determination of liver PEPCK activity and liver PEPCK mRNA abundance, and a glucose tolerance test were performed at 4 hours in fed rats and at 6 hours in fasted rats. LPS induced hypoglycemia (1.62 +/- 0.33 mmol/L, P < .05) at 6 hours in group 2 (fed-LPS), but not in group 4 (fasted-LPS). Hyperinsulinemia was not observed in either group 2 (fed-LPS) or group 4 (fasted-LPS). In group 2 (fed-LPS), liver gluconeogenesis decreased (3.0 +/- 0.3 mg/g liver, P < .01). PEPCK activity decreased from 0.65 +/- 0.07 (group 1) to 0.23 +/- 0.02 U (P < .01). PEPCK mRNA abundance also decreased from 100% +/- 10% to 40% +/- 10%. The glucose disappearance rate (t1/2) increased (P < .05) in group 2 (fed-LPS) and group 4 (fasted-LPS).(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.

Some enzymes of gluconeogenesis in various fractions of sarcocysts of Sarcocystis fusiformis of buffalo (Bubalus bubalis).

A biochemical study was conducted to assess the relative presence of some enzymes of gluconeogenesis in various fractions--the cyst wall, cyst fluid and zoites--of sarcocysts of Sarcocystis fusiformis obtained from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis). The activities of fructose 1,6-diphosphatase and malic enzyme were beyond detectable limits. Phosphoenol pyruvate carboxykinase (PEPCK) activity was maximally present in the zoites, whereas glucose 6-phosphatase activity was highest in the cyst wall. PEPCK seemed to play a crucial role in carbon dioxide fixation metabolism.

Hepatic lobular patterns of phosphoenolpyruvate carboxykinase, glycogen synthase, and glycogen phosphorylase in fasted and fed rats.

The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase (GS), and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat sections of tissue were collected on slides, fixed by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was confirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed animal but often showed a centrilobular pattern, and fasting did not alter the lobular distribution of either enzyme. Western blot analysis revealed no changes in the amount of these enzymes in the fed or fasted state. The cellular distribution of the three enzymes is similar to that of hepatic glycogen, in that the immunoreactive material has a clumped appearance in the periportal hepatocytes and is more dispersed in the pericentral cells. On fasting the periportal hepatocytes lose the dense compact localization of the enzymes and the protein becomes more homogeneously distributed throughout the cytosol. Further studies are needed to elucidate the functional significance of the regional heterogeneity of the glycogen-metabolizing enzymes and the molecular mechanisms regulating their gene expression.

Identification of reactive vicinal cysteines in Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases.

Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide. Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated. The fluorescence spectra of the labeled proteins showed the pyrene excimer emission band. The pyrenyl-derivatized enzymes were digested with trypsin after carboxymethylation, and two labeled peptides were isolated for each carboxykinase upon reverse-phase high-performance liquid chromatography. Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzyme), and cysteines 288 and 413 (rat enzyme) were labeled with the fluorescence SH-specific reagent. The relative reactivity of these residues was characterized. Labeling experiments utilizing the 5,5'-dithiobis(2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-groups occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region.

Effect of glucose and insulin deprivation on differentiation and carbohydrate metabolism of rabbit proximal tubular cells in primary culture.

Rabbit proximal tubule cells in primary culture revert from gluconeogenesis to glycolysis. To determine whether glucose and insulin deprivation of the culture medium could prevent this metabolic conversion without a loss of differentiation, rabbit proximal tubule cells were cultured in hormonally defined medium free of glucose and insulin and compared to rabbit proximal tubule cells cultured in medium supplemented with 17.5 mM glucose and 5 micrograms/ml insulin. In the two culture conditions, RPT cells grew at a similar rate and reached confluency within 4-5 days. Patterns of enzyme activity, including brush-border hydrolases, N-acetyl-beta-D-glucosaminidase and glutathione-S-transferases as a function of culture time were comparable in the two media. During the growth phase in glucose- and insulin-free medium, cells showed higher sodium-dependent glucose uptake. Scanning electron microscopy revealed a high density of microvilli at confluency regardless of the culture conditions. In both the presence and absence of glucose and insulin, the activities of gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase, as well as basal and pyruvate-stimulated glucose production fell markedly as a function of time. By contrast, glucose and insulin deprivation greatly reduced both the lactate production rate and the activities of glycolytic enzymes, pyruvate kinase, hexokinase and lactate dehydrogenase.

The effects of changes in the definitive host environment on the metabolism of Hymenolepis diminuta during growth and maturation.

Flexibility in the metabolism of Hymenolepis diminuta is associated with changing intrinsic requirements during maturation but is also influenced by extrinsic factors, that is, by the nature of the host environment. End-products of carbohydrate metabolism and enzyme activities in worm extracts were used as indicators of metabolic regulation in H. diminuta recovered at various times postinfection. The predominant end-product from 6-day-old worms is lactate, generated by cytosolic glycolysis. As the cestode matures in the host, lactate production by the whole worm decreases and greater amounts of the mitochondrial end-products, succinate and acetate, are detected. A stable, dichotomous carbon flow to lactate, succinate and acetate is observed from 12 days post-infection. A metabolic gradient along the length of individual strobila is also evident. It extends from glycolysis, in the anterior region, to mitochondrial dismutation in the posterior region. The transition from cytosolic to mitochondrial pathways during maturation and along the strobilus is delayed or suppressed in worms recovered from immunosensitized hosts. Four host environments were compared: unsensitized rats, rats immunosensitized with a primary infection of H. diminuta, rats immunosensitized with a primary infection of Nippostrongylus brasiliensis and mice concurrently infected with Heligmosomoides polygyrus. The specific activities of PK and PEPCK in whole worm extracts were similar in 10-, 21- and 35-day-old worms and did not differ in worms isolated from different host environments. However, the PEPCK/PK ratio is high in worms that utilize mitochondrial pathways and low in worms that produce predominantly lactate. LDH activity is high in lactate producers. It is concluded that the pattern of metabolism in H. diminuta is influenced by many effectors in the host environment.

Identification of vicinal thiols of phosphoenolpyruvate carboxykinase (GTP).

Phosphoenolpyruvate carboxykinase (PEPCK) from the cytosol of rat liver has 13 cysteines, at least one of which (Cys288) is known to be very reactive and critical for catalytic activity (Lewis, C. T., Seyer, J. M., and Carlson, G. M. (1989) J. Biol. Chem. 264, 27-33). Previous results provided evidence for the existence of at least 1 pair of vicinal cysteines within or near the active site of PEPCK (Lewis, C. T., Haley, B. E., and Carlson, G. M. (1989) Biochemistry 28, 9248-9255). An intramolecular cystine disulfide is induced to form upon treatment of PEPCK with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) or upon irradiation of the enzyme in the presence of the photoaffinity probe 8-azidoGTP. In each case, modification is accompanied by a substantial loss in catalytic activity, and substrates protect against inactivation and modification. We now report the identification of these modified thiols by differential alkylation of cysteines and half-cystines with radioactive iodoacetate, followed by isolation and sequencing of the modified tryptic peptides. The results indicate that the disulfide formed by equimolar Nbs2 lies within a 15-residue region of the PEPCK sequence that includes Cys399, Cys407, and Cys413. In addition, Cys407 and/or Cys413 also appear to participate in formation of the disulfide induced by 8-azidoGTP. These thiols lie very near a consensus sequence that has been suggested to represent the binding site for the guanine ring of GTP.