Rate Of Glucose Synthesis Research Articles

Phosphoenolpyruvate carboxykinase revisited: Insights into its metabolic role

Phosphoenolpyruvate carboxykinase revisited: Insights into its metabolic role

Increased glucose synthesis in renal tubule fragments from hyperthyroid rats

Rates of glucose synthesis from several substrates were examined in renal tubule fragments from hyperthyroid rats. A hyperthyroid state was induced by daily intraperitoneal injections of thyroxine (T4) (100 µg/100 g body weight) for 14 days. At the end of the experimental period, plasma triiodothyronine and T4levels were six and eight times higher, respectively, than initial values. Hyperthyroid rats gained less weight and had lower blood glucose despite an increased food intake. In both control and hyperthyroid rats, rates of glucose production by renal tubule fragments were higher with glutamine and glycerol than with lactate, alanine, or glutamate. T4treatment induced a significant increase in the de novo glucose synthesis from all substrates, except glutamine. The highest percent increase was obtained with alanine (64%), compared with 31-40% for glutamate, lactate, and glycerol. The T4treatment induced increase in glucose synthesis by renal tubule fragments suggests that renal gluconeogenesis contributes to enhance glucose production in hyperthyroidism.Key words: hyperthyroidism, rat, renal gluconeogenesis, renal tubule fragments, glutamate, glutamine, lactate, alanine, glycerol.

Effect of lactation on gluconeogenesis and ketogenesis in ovine hepatocytes

1. 1. Rates of glucose synthesis from radioactive precursors and ketogenesis were determined in hepatocytes from control and lactating sheep. 2. 2. Gluconeogenesis from propionate was the same in both groups. Gluconeogenesis from lactate + pyruvate was three-fold higher in hepatocytes from lactating sheep. Palmitate stimulated gluconeogenesis from lactate + pyruvate in both groups. 3. 3. Rates of ketogenesis from palmitate but not butyrate were slightly higher in hepatocytes from lactating sheep. No other differences in the metabolism of palmitate or butyrate were seen in the two groups. Exogenous carnitine stimulated ketogenesis from palmitate. Propionate inhibited ketogenesis from palmitate and butyrate. Lactate + pyruvate also inhibited ketogenesis slightly but stimulated oxidation and esterification. 4. 4. It is concluded that the major changes in glucose and ketone production seen in the lactating ruminant are not the result of long-term changes within the hepatocyte but occur because of the changes in substrate supply to the liver and changes in intracellular concentrations of metabolites.

Mitochondrial carbonic anhydrase is involved in rat renal glucose synthesis

At 37 degrees C, pH 7.4, carbonic anhydrase activity (kenz) of disrupted rat renal proximal tubules and cortical mitochondria was 2.5 +/- 0.8 (n = 3) and 0.15 +/- 0.40 (n = 3) ml.mg-1.s-1, respectively. Turnover number for renal mitochondrial carbonic anhydrase (CA V) was 24,000 s-1. CA V activity of intact mitochondria was completely inhibited by 0.15 microM ethoxzolamide (EZ). Intact proximal tubules, prepared from 48-h starved male rats, were incubated at 37 degrees C in 10 mM pyruvate in Krebs-Henseleit bicarbonate saline buffer, 5% CO2-95% O2. The rate of glucose synthesis over 60 min was reduced 50% by including 0.6 microM EZ in the incubation solution. The concentration of NaHCO3 was doubled to 50 mM (with a corresponding decrease in NaCl) and the solution gassed with 10% CO2-90% O2; 2.4 microM EZ no longer decreased glucose synthesis. It was concluded that inhibition of glucose synthesis by EZ was directly a result of inhibiting the carbonic anhydrases. The rate of glucose production was subsequently determined with tubules incubating in a HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) buffer; this rate was decreased 50% by 0.6 microM EZ. These data support the hypotheses that CA V provides HCO3- for pyruvate carboxylase and that CO2 can be provided by tubular metabolism. Intact tubules were incubated in from 5 to 20 mM pyruvate in either 25 or 50 mM HCO3-; in either buffer, the rate of glucose synthesis was similar, increasing with increasing pyruvate concentration. At no pyruvate concentration was there a change in the rate of glucose production when tubules were incubated in 50 mM HCO3- buffer with 1.6 microM EZ. These data also support the hypothesis that CA V provides the HCO3- substrate for pyruvate carboxylation when there is a high rate of intracellular CO2 production and external CO2 is low. It is further concluded that the cytosolic carbonic anhydrase (CA II) and the membrane-bound carbonic anhydrase (CA IV) are not involved in glucose synthesis from pyruvate.

“Hormone-like” effect of adenosine and inosine on gluconeogenesis from lactate in isolated hepatocytes

In isolated rat hepatocytes adenosine and inosine showed a dose-dependent increase in the rate of glucose synthesis from lactate with a K a of 7.5 x 10 −8 and 9 x 10 −8 M, respectively. Absence of this action was recorded with: IMP, xanthosine, adenine, hypoxanthine, and uric acid. A reciprocal inhibition of individual gluconeogenic stimulation was found in cells incubated with glucagon or epinephrine and adenosine, but not with inosine. 5'−(N-ethyl) carboxamido adenosine was more potent than adenosine, whereas N 6-(L-2- phenylisopropyl)-adenosine antagonized the stimulation of gluconeogenesis by adenosine. Neither of the analogs used modified the stimulatory role of inosine on the studied pathway. Adenosine and inosine may be involved in the short term regulation of gluconeogenesis.

Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate-limiting. This stimulation was also observed when the activities of phospho enolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force-flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.

Effect of cholera enterotoxin on carbohydrate metabolism in the liver and small intestinal mucosa of rabbits

Changes in carbohydrate metabolism were studied in the isolated intestinal loops of rabbits during secretory diarrhea, induced by cholera enterotoxin. Glucose synthesis level in the small intestinal mucosa and liver was measured by isotope technique, using L-alanine as a precursor. Intestinal gluconeogenesis, calculated per mg of protein, appeared to be twice higher than in the liver of fasting rabbits. Cholera enterotoxin administration enhanced gluconeogenesis in the liver by 60%, as compared to the control. The rate of glucose synthesis and glucose-6-phosphatase activity in the intestinal mucosa remained unchanged, whereas glucose-6-phosphatase in the liver was slightly inhibited. It is suggested that gluconeogenesis in the liver supplies glucose as a convenient energy source for the secretory process induced by cholera enterotoxin in the rabbit small intestine.

Kidney gluconeogenesis: Its importance to net glucose synthesis during the development of chick embryos

1. 1. A technique is described for the preparation of viable isolated kidney tubules from chick embryos. 2. 2. Assessment of the potencies of various possible gluconeogenic precursors indicates that kidney is the site of net glucose synthesis from physiologically relevant precursors during embryonic life. 3. 3. The plasma concentrations of physiological gluconeogenic precursors was determined and the rates of glucose synthesis by tubules was measured from a precursor mixture which simulated plasma contents.

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with l-buthionine sulfoximine, a specific inhibitor of γ-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 μmol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH GSSG ratio remained unchanged in all cases.

Effect of exogenous glucagon and free fatty acids on gluconeogenesis in fasting neonatal pigs.

Thirty-two pigs (1 d old) were used to determine if exogenous glucagon and(or) free fatty acids (FFA oleic acid) would enhance gluconeogenesis and glucose homeostasis during fasting. Pigs were acquired at birth, fitted with an indwelling arterial cannula (via umbilicus) and fasted 24 h to deplete liver glycogen. A jugular cannula was inserted nonsurgically 8 to 10 h before initiation of a primed-continuous infusion consisting of control (excipient), glucagon (Glu), oleic acid (FFA), or both glucagon and oleic acid (Glu-FFA). Plasma Glu averaged 395 pg/ml preinfusion and was similar across treatments. The concentration increased fivefold (P less than .05) by 80 min for Glu and Glu-FFA pigs and remained constant thereafter (160 min: 2,379, 2,258 pg/ml; 240 min: 2,355, 2,274 pg/ml, respectively). Glucagon infusion did not alter plasma glucose after 240 min of infusion (control, 50 vs Glu, 51 mg/dl); however, Glu-FFA effected an increase (60 mg/dl, P less than .10). In contrast, pigs infused with FFA alone had a lower glucose concentration (40 mg/dl, P less than .10). Rate of glucose synthesis was determined using liver slices, acquired immediately postinfusion, with alanine and lactate as substrate (7.5 mM). The rate of synthesis was not altered by Glu or Glu-FFA infusion (2.91, 2.43 mumol glucose X g-1 X h-1 vs 2.91 for control). In contrast, exogenous FFA reduced synthesis to 1.85 mumol glucose X g-1 X h-1 (P less than .05) with lactate as substrate. It appears that Glu is not the primary factor limiting gluconeogenesis in fasting newborn pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary culture of the freshly isolated liver cells of the eel.

The primary culture of the freshly isolated liver cells of the eel was carried out. The isolated cells could attach, spread and form monolayer only on the dish which was coated with fibronectin. The cell number decreased during the culture and decreased by about 40 to 50% of the initial cell number after 7 days culture. The rate of protein synthesis increased during 8 days culture and the synthesis of the intracellular proteins was always higher than that of the extracellular proteins during the same period. From the investigation of synthesis of the extracellular proteins by fluorography a few proteins were synthesized and excreted by cultured hepatocytes, but the synthesis of the proteins corresponding to the bovine serum albumin was not observed.From the comparison of glucose, glycogen, and protein synthesis in the freshly isolated and cultured cells, it was found that the cultured cells had higher activity of glycogen and protein synthesis than the isolated cells. The rate of protein synthesis in the cultured cells was 10 to 100 times higher than that in the isolated cells. However, the rate of glucose synthesis in the freshly isolated liver cells was higher than that of the cultured cells.

GLUCOSE METABOLISM IN FAT AND THIN ADULT SHEEP

The effect of body fatness on glucose metabolism was studied in six adult crossbred wethers divided into two groups weighing 90 kg (fat) or 55 kg (thin). Glucose turnover measurements were made using 6-3H-glucose by single injection at four levels of metabolizable energy intake: high, 17 MJ/day; intermediate, 10 MJ/day; low, 6 MJ/day; and fasting for both thin and fat groups. The greatest group differences occurred at the high feeding level. Body pool size and glucose space were significantly lower (P < 0.001) for the fat sheep and were reduced at fasting for both fatness levels. The mean irreversible loss rate was not affected by body fatness but was significantly affected (P < 0.001) by feeding level. Glucose recycled as a proportion of total entry rate was significantly affected by body fatness and was dependent on a positive or negative energy balance. When in negative energy balance the sheep maintained blood glucose within the normal range by recycling up to four times more glucose than was utilized. It appears that rate of glucose synthesis is related to energy balance and metabolizable energy intake. A tendency toward insulin insensitivity in the fat sheep could be related to high levels of recycling, and a greater glucose drain or prolonged fast could be more serious with the decreased glucose pool size and space. Key words: Glucose metabolism, ruminant, obese, energy balance

Rate of gluconeogenesis in the liver of rats with myocardial infarction

It has been shown for the first time that the rate of glucose synthesis from endogenous non-carbohydrate compounds in the perfused liver of rats with occlusion of the descending branch of the left coronary artery increased. Gluconeogenesis appeared to be the most intense by the third day after occlusion. The rate of gluconeogenesis was identical both in control and experimental animals by the 10th and 20th days after occlusion.

Lactate-stimulated ethanol oxidation in isolated rat hepatocytes.

1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8-0.9mumol/min per g wet wt. of cells. Addition of 10mm-lactate increased this rate 2-fold. 2. Quinolinate (5mm) or tryptophan (1mm) decreased the rate of gluconeogenesis with 10mm-lactate and 8mm-ethanol from 0.39 to 0.04-0.08mumol/min per g wet wt. of cells, but rates of ethanol oxidation were not decreased. From these results it appears that acceleration of ethanol oxidation by lactate is not dependent upon the stimulation of gluconeogenesis and the consequent increased demand for ATP. 3. As another test of the relationship between ethanol oxidation and gluconeogenesis, the initial lactate concentration was varied from 0.5mm to 10mm and pyruvate was added to give an initial [lactate]/[pyruvate] ratio of 10. This substrate combination gave a large stimulation of ethanol oxidation (from 0.8 to 2.6mumol/min per g wet wt. of cells) at low lactate concentrations (0.5-2.0mm), but rates remained nearly constant (2.6-3.0mumol/min per g wet wt. of cells) at higher lactate concentrations (2.0-10mm). 4. In contrast, owing to the presence of ethanol, the rate of glucose synthesis was only slightly increased (from 0.08 to 0.12mumol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65mumol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O(2) uptake increased with increasing substrate concentration over the entire range. 6. Changes in concentrations of glutamate and 2-oxoglutarate closely paralleled changes in the rate of ethanol oxidation. 7. In isolated hepatocytes, rates of ethanol oxidation are lower than those in vivo apparently because of depletion of malate-aspartate shuttle intermediates during cell preparation. Rates are returned to those observed in vivo by substrates that increase the intracellular concentration of shuttle metabolites.

Effects of Trichostrongylus colobriformis (Nematoda) on the nutrition and metabolism of sheep. IV.* Glucose metabolism

The effect of infection with Trichostrongylus co!ubriformis on quantitative aspects of glucose metabolism was studied in 12 Merino lambs. Total entry rate, rate of irreversible loss and rate of recycling were measured simultaneously by means of [U-14C]glucose and [3-3H]glucose. The feed intake of all sheep was maintained at 540 g/day throughout the experiment. The total entry rate of glucose in infected sheep was greater than in their controls. There was a smaller difference in the irreversible loss of glucose because of increased recycling of glucose carbon in infected sheep. It was concluded that the rate of glucose synthesis was higher in infected sheep compared with control sheep on the same feed intake, because there was more recycling and possibly increased availability of propionate from the large intestine. However, this increase would not compensate for the reduced intake of glucose precursors by infected sheep fed ad libitum, so the overall effect of T. colubriformis infection would be to reduce glucose synthesis. ___________________ *Part III, Aust. J. Agric. Res., 28: 155 (1977).

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.

Use of endogenous triglycerides to support gluconeogenesis in the perfused isolated rat liver.

The carbon balances in isolated perfused rat liver during gluconeogenesis from L-alanine and sodium L-lactate indicate that assuming the substrate unaccounted for were fully oxidized the energy yielded was not sufficient to support the observed rates of glucose synthesis. This observation indicates that endogenous substrates must also be oxidized. The possibility that endogenous fatty acid oxidation was the source of the energy needed to support glucose synthesis was investigated by measuring the rate of 14CO2 formation from tracer quantities of added [U-14C] palmitate. Short pulses of L-alanine or sodium L-lactate infusion produced an increased rate of 14CO2 production paralleled by increases in oxygen uptake indicating that more endogenous fuel is being mobilized. That the rate of 14CO2 output is an expression of fatty acid mobilization was supported by experiments demonstrating that the addition of octanoate to dilute the fatty acid pool produced an immediate fall in the rate of 14CO2 output. On the other hand, the administration of glucose produced no changes in oxygen uptake or 14CO2 output. However, lactate even in the presence of glucose induced a rise in 14CO2 production which occurred in parallel with the enhancement in oxygen uptake. It is concluded that mobilization of hepatic endogenous fatty acid is a metabolic event intimately associated with enhancement of gluconeogenesis. Consequently the control of the different steps of this process may indirectly control gluconeogenesis.

Glucose formation by the R3230AC rat mammary adenocarcinoma

1. 1. The presence of d-fructose-1,6-diphosphate 1-phosphohydrolase (E.C.3.1.3.11) activity has been established in the mammary glands of late pregnant Fisher 344 rats and in the R3230AC rat mammary adenocarcinoma. 2. 2. Homogenates of the R3230AC tumour are capable of synthesizing glucose from fructose-1,6-diphosphate a and other gluconeogenic precursors. The rate of glucose synthesis from fructose-1,6-diphosphate is decreased by high substrate concentrations, estrogen administration to the tumour bearing rats and by the addition of 5′AMP or ATP to the incubation mixture. 3. 3. Starvation increases glucose formation from fructose-1,6-diphosphate in the estrogen-treated rats but has no effect in the non-treated animals. 4. 4. Tumour homogenates produced α-glycerophosphate from glucose and other glycolytic precursors. The latter finding points to the presence of the α-glycerophosphate shunt in the R3230AC adenocarcinoma.

The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells.

The possibility that hormones control hepatic gluconeogenesis via the regulation of the rate of mitochondrial pyruvate carboxylation was investigated with the use of suspensions of liver cells isolated from fasted rats. The mitochondria prepared from liver cells were judged in good condition as they exhibited satisfactory phosphorus-oxygen and respiratory control ratios and transported Ca2+ and K+ ions in an energy-dependent manner. Addition of glucagon, epinephrine, or cyclic adenosine 3':5'-monophosphate to liver cells caused a 50 to 80% increase in the rate of glucose synthesis from lactate. When mitochondria were isolated from the cells after treatment with these agonists, they displayed 2- to 3-fold increases in the rate of pyruvate carboxylation, pyruvate decarboxylation, and pyruvate uptake. These mitochondrial changes are similar to those obtained in hepatic mitochondria prepared from intact, hormone-treated rats. The mitochondrial responses were specific for agents that stimulated gluconeogenesis; no response occurred with 5'-AMP or cyclic adenosine 2':3'-monophosphate. In the cell suspensions, the dose response curves for the activation of mitochondrial pyruvate metabolism and for increased glucose synthesis from L-lactate were coincident with four different agonists. The mitochondrial changes resulting from stimulation with glucagon developed in 1 to 2 min after the rise in cyclic adenosine 3':5'-monophosphate and occurred at least as early as the increase in the rate of gluconeogenesis. When the intracellular level of cyclic adenosine 3':5'-monophosphate returned to basal values, the rates of mitochondrial pyruvate carboxylation and glucose synthesis also declined to control levels. It is concluded that the rate of mitochondrial pyruvate metabolisms can be increased by hormones and cyclic nucleotides and that control of mitochondrial pyruvate carboxylation is an important regulatory site of hepatic gluconeogenesis.

Effect of hydrocortisone and acute hypoxia on gluconeogenesis in the rat liver

Acute hypoxic hypoxia leads to increased glucose formation by rat liver slices from pyruvate,α-glycerophosphate,α-ketoglutarate, and fructose. During exposure to hypoxia after prolonged administration of hydrocortisone acetate, increasing gluconeogenesis from the above-mentioned substances and also from alanine, lactate, and glycerol, the rate of glucose synthesis in liver slices was higher than in the control but lower than after the action of hydrocortisone alone.