Liver Phosphoenolpyruvate Carboxykinase Research Articles

Enzymes of carbohydrate metabolism in young and adult rats fed diets differing in fat and carbohydrate.

Glycogen content as well as glycolytic, gluconeogenic and fatty acid synthesis enzyme activities were monitored in young and adult male rats fed diets differing in fat content: 11% (low), 22% (medium) and 42% (high) of total energy from fat. The results showed significant differences in the responses of young and adult rats to changes in dietary fat and carbohydrate. In young animals, increasing dietary fat decreased total liver glycogen phosphorylase (GP), pyruvate kinase (PK), glycerol 3-phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme (ME), ATP-citrate lyase (ATP-CL) and fatty acid synthase (FAS). Increasing dietary fat also affected enzyme levels in other tissues: hexokinase (HK) and pyruvate dehydrogenase (PDH) activities decreased whereas skeletal muscle PK activity increased. The pattern of enzyme changes was similar in livers of fed adults with the exception that liver GP was not affected by dietary manipulations. Overnight food deprivation decreased liver glucokinase (GK), ME, ATP-CL, and FAS activities and increased liver phosphoenolpyruvate carboxykinase (PEPCK) and phosphofructokinase in both young and adult animals. In young animals, food deprivation also: (i) reduced liver GK and PK, (ii) increased kidney PEPCK, (iii) decreased muscle PEPCK and (iv) decreased kidney PDH. Food-deprived adults had increased skeletal muscle PEPCK and kidney glycogen synthetase as well as decreased kidney PEPCK muscle GP activity. These differences suggest that young animals are somewhat more responsive to changes in dietary manipulations. They also show that overnight food restriction causes a more profound metabolic re-organization in younger than in older animals.

Developmental Changes in the Phospho(enol)pyruvate Carboxykinase Gene Expression in Small Intestine and Liver of Suckling Rats

The cytosolic enzyme phospho(enol)pyruvate carboxykinase (PEPCK) is markedly expressed in the intestinal mucosa of suckling rats. The expression is located in the small intestine, but there is no expression in stomach, colon, or cecum. The expression changes with age. The mRNA levels at birth are very low, increase after the first lactation, reach maximum levels between 3 and 9 days after birth, and then decrease smoothly. At weaning, when animals begin to feed on a solid chow diet, the expression falls to adult levels, which are hardly detectable. Mother's milk may influence the intestinal expression, since in rats weaned at Day 18, 3 days before normal weaning, the mRNA levels decreased dramatically. mRNA levels for PEPCK in liver present a rather different developmental pattern from that of intestine, remaining high at weaning and in adult rats. On the ninth day after birth, the mRNA levels are the same in intestine and liver.

INSULIN-LIKE GROWTH FACTOR-1 LOWERS mRNA LEVEL OF PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) GENE, BUT DOES NOT SUPPRESS AMYLIN GENE EXPRESSION IN NEWBORN DOGS ▴ 1836

Insulin-like growth factor-1 (IGF-1) exerts insulin-like effects on glucose homeostasis. Amylin is a newly discovered pancreatic hormone which may impair insulin action. IGF-1 has been reported to reduce plasma amylin level in adult rats. In the present study, the effects of IGF-1 on the expression of the liver phosphoenolpyruvate carboxykinase (PEPCK) gene and the pancreatic amylin gene in newborn dogs were tested to determine the effects on neonatal glucose metabolism by IGF-1. After C-section delivery, euglycemic hyper-IGF-1 clamps(IGF-1 infusion rate: 10 μg/kg/min) were performed in newborn dogs (N=3). Total RNAs were isolated from frozen liver and pancreas. Northern blottings were performed using a rat PEPCK cDNA probe and a dog amylin cDNA probe to measure the mRNA levels of the PEPCK and amylin genes. The mRNA bands were quantitatively analyzed by a densitometer. The plasma concentration of amylin and IGF-1 were analyzed by radioimmunoassay. Results: 1) After the clamps, the plasma IGF-1 concentration was sharply increased to 2,822.2± 539.5 ng/ml from 128.3 ± 15.4 ng/ml (basal), whereas the plasma glucose level (143.0 ± 13.7 mg/dl) was kept at basal level(138.3 ± 18.1) by a variable infusion of glucose; 2) IGF-1 lowered the liver PEPCK mRNA level (75.1 ± 2.8) by 45.7%, compared to control(138.4 ± 41.2%); 3) IGF-1 inhibited neither amylin mRNA level (106.7± 14.2%) compared with the basal (100.0 ± 5.9%) nor the plasma amylin level (56.6 ± 3.8 pg/ml) compared to the basal (60.3 ± 5.5 pg/ml). The results suggest that IGF-1 may effectively suppress neonatal gluconeogenesis but that the unaffected amylin expression in newborn dogs by IGF-1 may be a counterregulatory factor for inducing insulin resistance in neonatal mammalians.

Glycerol synthesis in the rainbow smelt Osmerus mordax

Rainbow smelt, Osmerus mordax, maintain high glycerol levels in winter to avoid freezing. After intramuscular injection of 14C-labeled glucose, [14C]glycerol was found in the blood, liver and muscle, indicating that glycogen is a source of glycerol. Levels of both the active and inactive forms of glycogen phosphorylase were higher in muscle in winter than in autumn, although the fraction in the active form did not change significantly. More of the phosphorylase was in the active form in the liver than in the muscle. Short-term starvation resulted in a significant decrease in the level of glycogen soon after the stomachs were emptied, presumably to replace glycerol lost to the water. However, tissue glycerol levels remained relatively high, despite a near depletion of glycogen reserves. Triglyceride levels increased slightly during starvation, indicating that triglycerides were not involved in glycerol synthesis. After intramuscular injection of 14C-labeled pyruvate, [14C]glycerol was found in the blood, liver and muscle, indicating a second route, presumably from muscle protein, to glycerol synthesis. Liver phosphoenolpyruvate carboxykinase activity was slightly higher in winter, possibly to assist in the conversion of pyruvate to glycerol.

Glucokinase and cytosolic phosphoenolpyruvate carboxykinase (GTP) in the human liver. Regulation of gene expression in cultured hepatocytes.

Glucokinase and phosphoenolpyruvate carboxykinase are key enzymes of glucose metabolism in the rat liver. The former is considered to be instrumental in regulating glucose hepatic release/uptake according to the glycaemia level, and cytosolic phosphoenolpyruvate carboxykinase is a major flux-generating enzyme for gluconeogenesis. The level of expression of both enzymes and the regulation of their mRNAs in the human liver cell were investigated. Surgical biopsies of liver from patients undergoing partial hepatectomies and parenchymal hepatocytes derived from the biopsies were used to assay glucokinase, hexokinase and phosphoenolpyruvate carboxykinase activities. Hepatocytes were placed in culture and the actions of insulin, glucagon and cAMP on glucokinase and phosphoenolpyruvate carboxykinase mRNAs were studied. The main results are: (a) glucokinase accounts for 95% of the glucose phosphorylation activity of human hepatocytes, although this fact is masked in assays of total liver tissue; (b) glucokinase activity is set at a lower level in human hepatocytes than in rat hepatocytes, and vice-versa for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase; and (c) as previously shown in rat liver, glucokinase and phosphoenolpyruvate carboxykinase mRNAs are regulated in a reciprocal fashion in human hepatocytes, insulin inducing the first enzyme and repressing the latter, whereas glucagon has opposite effects. These data have interesting implications with respect to metabolic regulation and intracellular hormone signaling in the human liver.

Correlation between toxicity and effects on intermediary metabolism in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated male C57BL/6J and DBA/2J mice.

Male mice were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gavage. C57BL/6J (C57) mice received 0.03 to 235 micrograms/kg, DBA/2J (DBA) mice 1 to 3295 micrograms/kg. On Day 8 after dosing blood was collected, and livers and kidneys were removed. Body weights and feed intake were not much affected until Day 8 after exposure. Hepatomegaly developed at doses above 3 and 97.5 micrograms/kg in C57 and DBA mice, respectively. Ethoxyresorufin O-deethylase activity was induced in liver with an ED50 of 1.1 and 16 micrograms/kg and in kidney with an ED50 of 65 and 380 micrograms/kg in C57 and DBA mice, respectively. The activity of phosphoenolpyruvate carboxykinase (PEPCK) in livers of both mouse strains was reduced over the entire dose range, displaying a plateau in the dose response at the onset of acute toxicity of TCDD. This enzyme activity was decreased by as much as 80% at the respective lethal doses. PEPCK activity in kidney was not affected. Glucose-6-phosphatase activity (G-6-Pase) in liver was altered only in the lethal dose range with a maximum reduction of about 50%. Serum glucose concentration was reduced over the entire dose range, but the reduction was significant only at doses in which G-6-Pase activity was affected, reaching levels as low as 3 mmol/liter in DBA mice. Tryptophan 2,3-dioxygenase activity was not lowered at any dose of TCDD in either mouse strain, and no increase in serum tryptophan levels was observed. Serum levels of thyroxine (T4) and triiodothyronine (T3) were dose dependently decreased over most of the dose range administered, with T3 levels exactly paralleling T4 levels in both mouse strains. It is concluded that TCDD causes acute toxicity in male C57 and DBA mice by a severe reduction of gluconeogenesis, but, in contrast to rats, it does not affect tryptophan homeostasis. Following administration of TCDD serum T3 levels in the mouse appear to correlate with T4 levels, whereas in the rat they are independent of each other.

Subchronic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and their reversibility in male Sprague-Dawley rats

The hypothesis tested in this experiment is that effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) show identical dose-responses after subchronic as after acute exposure when the dose is corrected for toxicokinetics. Groups of male Sprague-Dawley (S-D) rats were administered orally a total dose of 0, 0.2, 2.3, 11.5, 35, 70 or 115 μg/kg of TCDD over a period of 10 weeks at 4 ml/kg of vehicle. Body weight was recorded weekly. One week after the last dose of TCDD one half of the rats was killed and tryptophan 2,3-dioxygenase (TdO), 7-ethoxyresorufin- O-deethylase (EROD) and phosphoenolpyruvate carboxykinase (PEPCK) activities were measured in livers, whereas tryptophan and total T4 (TT4) were determined in serum. The results show that the dose-responses for decreased TdO and PEPCK activity and elevated serum tryptophan levels are similar if not the same as the dose-response for subchronic retardation of body weight increase. They also demonstrate that the dose-responses for the induction of EROD activity and the reduction of serum TT4 occurred at much lower doses than those for decreased TdO and PEPCK activities or elevated tryptophan levels and mortality. After a 6-week recovery period, PEPCK and TdO activities in liver as well as tryptophan in serum returned to near control values, whereas EROD activity and serum TT4 still displayed a dose-dependent induction and reduction, respectively, albeit both shifted to the right in accordance with toxicokinetics. These data support the notion that subchronic dose-responses of TCDD are similar to acute dose-responses when corrected for toxicokinetics and that at least some TCDD-induced effects are reversible also in accordance with toxicokinetics.

Excessive glucose production, rather than insulin resistance, accounts for hyperglycaemia in recent-onset streptozotocin-diabetic rats

Glucose production and utilization and activities of key enzymes involved in liver and muscle glucose metabolism were studied in post-absorptive streptozotocin-diabetic rats after 12 h of severe hyperglycaemia (17.5±0.5 mmol/l) and insulinopenia (5±1 ΜU/ml). Basal glucose production was increased: 36.6±3.0 mg·kg·min−1, vs 24.4±2.5 in controls (p<0.05); liver glycogen concentration was decreased by 40% (p<0.05); liver phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities were increased by 375 and 156%, respectively (p<0.001 and <0.01). During a euglycaemic clamp at a plasma insulin level of 200 ΜU/ml, glucose production was totally suppressed in controls, but persisted at 20% of basal in diabetic rats. In these rats, glucose production was suppressed at a plasma insulin level of 2500 ΜU/ml. Basal whole body glucose utilization rate, 2-deoxy-1-[3H]-d-glucose ([3H]-2dG) uptake by muscles and muscle glycogen concentrations were similar in both groups, as well as total and active forms of pyruvate dehydrogenase and glycogen synthase activities. During the euglycaemic clamp, the total body glucose utilization rates and [3H]-2dG uptake by muscles were similar in control and diabetic rats at a plasma insulin level of 200 ΜU/ ml, but lower in diabetic rats at a plasma insulin level of 2500 ΜU/ml. We conclude 1) in recent-onset severely insulinopenic rats, an excessive glucose production via gluconeogenesis prevailed, mainly accounting for the concomitant hyperglycaemia. This excess glucose output cannot be attributed to liver insulin resistance: the gluconeogenic pathway is physiologically less sensitive than glycogenolysis to the inhibition by insulin. 2) Glucose utilization was apparently normal under hyperglycaemic conditions and at a lower insulin plateau of the euglycaemic clamp but suboptimal in the presence of maximal insulin concentrations, suggesting an early appearance of peripheral insulin resistance.

Effect of age on the expression of phosphoenolpyruvate carboxykinase in rat liver.

The effect of age on the expression of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) was studied using hepatocytes isolated from 6-, 15-, and 26-mo-old-male Fischer 344 rats fasted for 12 h. The activity of PEPCK in hepatocyte extracts decreased 40-50% (P < 0.05) between 6 and 26 mo of age. The age-related decrease in PEPCK activity was paralleled by a similar decrease (P < 0.05) in the level of PEPCK mRNA. Thus the decrease in PEPCK activity appears to arise from a decrease in level of the PEPCK transcript available for translation. The stability, i.e., half-life of PEPCK mRNA in hepatocytes isolated from 6- and 26-mo-old rats was similar; however, the nuclear transcription of PEPCK by hepatocytes isolated from 26-mo-old rats was 44% lower (P < 0.05) than the nuclear transcription of PEPCK by hepatocytes isolated from 6-mo-old rats. Thus the age-related decline in PEPCK mRNA levels in the liver of fasted rats appears to arise from a decrease in the transcription of PEPCK. A significant decrease (P < 0.05) in the activity and mRNA level of PEPCK also was observed with age in kidney tissue isolated from rats fasted for 12 h.

Involvement of liver and skeletal muscle in sucrose-induced insulin resistance: dose-response studies.

The ability of dietary sucrose to induce insulin resistance independent of changes in body weight is controversial. In the present study male rats were fed a high-starch (ST) diet (starch 68% of total kcal) ad libitum for 2 wk and then were fed equicalorically either the ST diet or a high-sucrose (SU) diet (sucrose 68% of total kcal) for 8 wk. Euglycemic, hyperinsulinemic (0, 1.2, 4.1, 8, 15 mU.kg-1.min-1, n = 6-8/group per dose) clamps were then used to establish dose-response relationships for glucose kinetics and metabolism. Body weight (513 +/- 3 g) and composition were similar between groups after the 8-wk dietary period. Glucose infusion rates (GIR; mg.kg-1.min-1) were significantly less in SU (0.9 +/- 5.8 +/- 0.6, 14.8 +/- 1.3, and 18 +/- 1.1) than in ST rats (4.1 +/- 0.9, 12.3 +/- 1.2, 22.6 +/- 1.5, and 25.9 +/- 1.8) at 1.2, 4.1, 8, and 15 mU.kg-1.min-1, respectively. Impaired suppression of endogenous glucose production accounted for 46, 43, 23, and 0% of the reduction in GIR in SU rats at 1.2, 4.1, 8, and 15 mU.kg-1.min-1, respectively. Despite basal hyperinsulinemia (38 +/- 2 microU/ml in SU vs. 26 +/- 2 microU/ml in ST rats), liver phosphoenolpyruvate carboxykinase (PEPCK) activity was 50% higher in SU than in ST rats and remained elevated in SU rats (by 30-40%) at the two lower insulin doses. No skeletal muscle glycogen accumulation occurred in SU rats at any of the insulin doses, and glycogen synthase I activity was significantly lower in SU rats at the two highest insulin doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Augmented glucoregulatory hormone concentrations during exhausting exercise in mildly iron-deficient rats

We hypothesized that augmented responses of glucoregulatory hormones in iron deficiency would enhance liver and muscle glycogenolysis, leading to increased gluconeogenic precursor (lactate) supply and upregulation of hepatic gluconeogenesis. Female weanling rats were randomly placed on either a mildly iron-deficient (-Fe; 15 mg Fe/kg diet) or an iron-sufficient (+Fe; 50 mg Fe/kg diet) diet for 4 wk and studied at rest and during exhaustive treadmill running. Hemoglobin was 9.0 +/- 0.2 and 13.1 +/- 0.3 g/dl in -Fe and +Fe, respectively, after 3.5 wk of dietary iron deficiency. Arterial plasma epinephrine (Epi), norepinephrine (NE), adrenocorticotropic hormone (ACTH), corticosterone, insulin, and glucagon levels were similar at rest in both groups, as were liver, gastrocnemius, and superficial and deep vastus medialis glycogen levels. Liver and kidney phosphoenolpyruvate carboxykinase (PEPCK) activities were similar in both groups. Maximum O2 consumption was decreased (22%) in -Fe. Respiratory exchange ratio (CO2 production/O2 consumption) was unaffected at rest but increased at maximum O2 consumption in -Fe. Time to exhaustion during a standardized running test (13.4 m/min, 0% grade) was decreased 45% in -Fe (63 +/- 5 vs. 116 +/- 10 min). During exercise, euglycemia was maintained in both groups, but blood lactate was elevated in -Fe. The mean net glycogen utilization during exercise was increased in liver (43%), soleus (33%), and superficial vastus medialis (106%) and decreased in the gastrocnemius (36%) in -Fe. Liver and kidney PEPCK activities were increased similarly at exhaustion in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Compartmentation of Liver Phosphoenolpyruvate Carboxykinase in the Aquatic Turtle Pseudemys Scripta Elegans: A Reassessment

ABSTRACT Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the conversion of oxaloacetate to pyruvate in the first step of gluconeogenesis. Since oxaloacetate is impermeable to the inner mitochondrial membrane, the localisation of PEPCK within the cell plays a major role in defining substrate preferences for gluconeogenesis. As a result, the immunochemically distinct isoenzymes of PEPCK found within the mitochondrial and the cytosolic cell fractions vary in their proportion to one another between organs and species and with prandial state.

Immunogold electron microscopic localization of phosphoenolpyruvate carboxykinase in rat liver

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting gluconeogenic enzyme which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. A gradient of PEPCK from periportal hepatocytes to pericentral hepatocytes has been demonstrated in normal rat liver; however, the subcellular distribution of PEPCK molecules within hepatocytes could not be determined from these light microscope studies.We have used two immunogold histochemical approaches to study the subcellar distribution of PEPCK in normal rat hepatocytes. In the first procedure 10μm cyrosections of 4% paraformaldehyde perfusion-fixed rat liver were collected on silanated slides and immunostained with goat anti-rat PEPCK (normal goat serum served as a control), and incubated in a secondary antibody (5nm gold-labeled rabbit anti-goat IgG) and a tertiary antibody (5nm gold-labeled goat anti-rabbit IgG). The immunogold stained samples, with or without silver enhancement (IGSS), were counterstained with pyronine Y, re-embedded in Visio- Bond and removed from the slides and semithin sectioned for epipolarized illumination combined with transmitted light microscopy (Figs. 1 and 2), or re-embedded in epoxy resin and sectioned for electron microscopy (EM, Fig. 4) In the second procedure 25, 50 and 100 μm free-floating vibratome sections of well fixed rat liver (4% paraformaldehyde plus 0.5% glutaraldehydeperfusion-fixed, immersion fixed overnight at 4°C) were immunogold stained using a 5nm gold labeled secondary antibody, with and without IGSS, osmicated, embedded in epoxy resin and sectioned for EM (Figure 3).

A physiological role of Mn2+ in the regulation of cytosolic phosphoenolpyruvate carboxykinase from rat liver is unlikely.

A cytosolic cell-free system prepared from rat liver was used to study the effect of bivalent cations on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Steady-state concentrations of oxaloacetate in the range 5-50 microM were generated from increasing concentrations of malate+fumarate (10:1); 2 mM ITP and 3 mM Mg2+ were added as cofactors. Micromolar concentrations of Mn2+, Fe2+ and, to a lesser extent, of Zn2+ and Co2+ were shown to stimulate PEPCK activity. Vmax. (mumol/min per g of liver) increased from 0.67 to 1.68 on addition of 5 microM Fe2+ and to 2.34 with 2 microM Mn2+, whereas no significant effect on the Km for oxaloacetate was observed. The apparent K(a) values (total) were 0.62 microM for Mn2+, 1.48 microM for Zn2+, 1.92 microM for Co2+ and 3.37 microM for Fe2+, being 2-8-fold lower than the corresponding published values. Variations of the free Mn2+ concentration were obtained (a) by increasing the Mn2+ concentration (i.e. activation curve) and (b) by simultaneous addition of Mn2+ and increasing concentrations of the chelating agent EGTA (i.e. inactivation curve). Different results were obtained for the activation and inactivation curves. The inactivation curve showed that PEPCK activity was almost unaffected by variations of the free Mn2+ concentration over the range 0.05-0.15 microM. Under comparable experimental conditions, rat liver arginase (another Mn(2+)-dependent enzyme) was completely inactivated. From kinetic evidence, the existence of two distinct molecular forms of cytosolic rat liver PEPCK with different Mn2+ affinities is postulated. Considering the high affinity of PEPCK for Mn2+ and its relative insensitivity to changes in the free Mn2+ concentration, it seems rather unlikely that changes in the free cation concentration play a major role in regulating PEPCK activity in vivo.

Induction of cytosolic phosphoenolpyruvate carboxykinase in the ovine fetal liver by chronic fetal hypoglycemia and hypoinsulinemia.

Fetal glucose production has been observed in the chronically hypoglycemic, hypoinsulinemic (HH) fetal lamb. The purpose of this study was to test the hypothesis that induction of hepatic gluconeogenic enzymes occurs in this condition. The activities of both mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase, and glucose-6-phosphatase, three key enzymes of gluconeogenesis, were determined in fetal sheep liver from HH lambs and controls (CONT). Pregnant ewes were maintained chronically hypoglycemic by continuous hyperinsulinemic clamps from approximately 80 d of gestational age (53% of gestation) for 6 wk. Fetuses (gestational age: HH = 136 +/- 2.6, CONT = 133 +/- 3.7 d) were maintained chronically hypoglycemic [HH = 0.51 +/- 0.05 versus CONT = 1.22 +/- 0.11 mmol/L (9.2 +/- 1.0 versus 21.9 +/- 11.9 mg/dL)] and hypoinsulinemic (HH = 3.3 +/- 0.6 versus CONT = 12.0 +/- 2.2 microU/mL) and delivered by cesarean section after measurement of fetal glucose production rate. Hepatic cytosolic PEPCK was 6.0 +/- 1.4 nmol/min/mg protein in CONT and 19.7 +/- 2.5 in HH lamb (p < 0.05), whereas mitochondrial PEPCK was not different between the two groups. Neither fructose-1,6-diphosphatase or glucose-6-phosphatase activities nor plasma glucagon levels were different between groups. These results suggest that chronic fetal hypoglycemia and hypoinsulinemia prematurely induce hepatic cytosolic PEPCK in the fetal lamb. The observed fetal glucose production in the HH fetal lamb may be due to gluconeogenesis.

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.

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is due to decreased mRNA levels

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 μg/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate thatthe decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.

Expression of the genes for the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase in avian liver during development.

Gluconeogenesis in the chicken has unique features due in part to the presence of two isozymes of PEPCK, a cytosolic form, PEPCK-C, and a mitochondrial form, PEPCK-M, which have novel patterns of expression. Here we show that, in contrast to mammals, in which PEPCK-C is not present in liver until after birth, avian PEPCK-C is expressed throughout embryonic life with mRNA levels gradually decreasing as development proceeds and becoming negligible at time of hatching. In addition two distinct mRNAs for PEPCK-M are expressed during development with specific patterns that vary among individual birds. These differences are likely to be genetic, as hormonal treatment of a chicken hepatoma cell line indicates that whereas the mRNA levels for PEPCK-C are hormonally regulated, the expression of PEPCK-M mRNA is unresponsive.

Human PCK1 encoding phosphoenolpyruvate carboxykinase is located on chromosome 20q13.2.

Cytoplasmic liver phosphoenolpyruvate carboxykinase (GTP) (PEPCK) catalyzes a rate-limiting step in gluconeogenesis. Primers derived from the rat liver PEPCK sequence were used to amplify a portion of the human liver cDNA and to screen a YAC library of human genomic DNA. The sequences of human and rat PEPCK cDNA differed at 16% of the nucleotides compared (45/291). Analysis of a human/rodent hybrid mapping panel demonstrated concordant segregation of PCK1 with chromosome 20. Fluorescence in situ hybridization with YAC DNA further localized PCK1 to subband 20q13.2.

Cell‐specific expression of cytosolic phosphoenolpyruvate carboxykinase in transgenic mice

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is expressed in multiple cell types in diverse tissues including liver, kidney, intestine, and white and brown adipose tissues. It can thus be considered a model system for examining the regulation of cell-specific transcription. The PEPCK gene is transcribed from a single start site, but studies of transgenic mice have revealed that distinct cis-acting elements (and thus different trans-acting factors) regulate PEPCK expression in hepatocytes, renal proximal tubule epithelial cells, and adipocytes. Hepatocytes require elements between -457 and +69 bp; renal proximal tubule epithelia require elements between -363 and +69 bp; and adipocytes require elements between -2086 and -888 bp. An additional element downstream of +69 bp is required to either attenuate PEPCK mRNA levels in liver and fat or increase renal PEPCK mRNA. We hypothesize that the transcription factors C/EBP and DBP are the principal tissue-specific regulators in liver, and that HNF-1 and perhaps C/EBP are important for kidney-specific PEPCK expression. We propose that the putative downstream element is involved in regulating PEPCK mRNA turnover in liver and fat. Finally, we suggest that the fat-specific element is an enhancer that requires a novel adipogenic regulatory factor, ARF6, to function. The long-term objective will be to fine map the cis-acting elements and identify the cognate trans-acting factors that regulate PEPCK in liver, kidney and fat. This information will help elucidate the combinatorial mechanisms that control the cell-specific expression of this complex gene.