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M Watford

Publications and source records attributed to M Watford.

At least 37 records · Page 2Linked to original sources
PubMed1993

Hepatic glutaminase expression: relationship to kidney-type glutaminase and to the urea cycle.

Glutamine functions as a major transport form of nitrogen and carbon within the body. In the liver, glutamine is hydrolyzed by a unique liver-type, phosphate-activated glutaminase, and the end products of hepatic glutamine catabolism are glucose and urea. Other tissues possess a different, kidney-type, glutaminase isozyme. The predicted amino acid sequences for the two glutaminases show a high degree of identity, indicating that they are products of different but related genes. Hepatic glutaminase activity is increased during diabetes, starvation, and on feeding high-protein diets, and decreased on feeding low-protein diets, whereas renal glutaminase appears to be regulated only by changes in acid-base status. Changes in the rate of gene transcription are the principal mechanism responsible for the long-term regulation of hepatic glutaminase, but the renal enzyme is regulated at the level of mRNA turnover. The pattern of regulation of hepatic glutaminase parallels that seen for genes encoding key enzymes of gluconeogenesis and urea synthesis, and indicates coordinate regulation of expression in keeping with the role of hepatic glutamine catabolism in these pathways.

Animals↗
PubMed1991

The urea cycle: a two-compartment system.

Channelling and enzyme localization remain controversial; they have been proposed for a number of metabolic pathways, especially glycolysis and the tricarboxylic acid cycle. One aspect that is often overlooked in discussions of channelling is that very tight channelling is readily accepted in pathways which occur in enzyme complexes, for example fatty acid synthetase, the 2-oxoacid dehydrogenase complexes and the protein synthesis/ribosome complex. As a metabolon the urea cycle is presently unique since it covers two conventional compartments. For the urea cycle, channelling appears to be almost complete with varying degrees of tightness at each step. Since a considerable portion of the nitrogen for urea synthesis is derived within the hepatocyte from amino acids, does this means that numerous enzyme-enzyme associations are required for this metabolon? Another important, as yet unaddressed, question is what are the consequences of channelling to theories of metabolic regulation? The answers will no doubt be forthcoming in the next few years as the concept of metabolons gains or loses acceptance.

Animals↗
PubMed1990

Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney.

Metabolic acidosis (6 days NH4Cl) causes a fourfold increase in the relative abundance of mRNA encoding phosphoenolpyruvate carboxykinase in rat kidney. Streptozotocin-diabetes (6 days) also results in an increased abundance of the mRNA but this increase can be prevented if the acidosis associated with bicarbonate is corrected by treatment with bicarbonate. The results confirm that renal phosphoenolpyruvate carboxykinase is regulated primarily by changes in acid-base status and that this control is at a pretranslational step. Isolated kidney tubules in short-term incubation have been used to identify which agents regulate levels of phosphoenolpyruvate carboxykinase mRNA. The relative abundance of the mRNA was increased by glucocorticoids and hormones which act via cAMP, or by cAMP analogues directly, but was not affected by hormones acting via Ca2+. Neither incubation at pH 7.1 nor the presence of serum from acidotic rats had any effect on the level of phosphoenolpyruvate carboxykinase mRNA. It is concluded that acidosis, glucocorticoids, and cAMP independently regulate expression of renal phosphoenolpyruvate carboxykinase.

Acid-Base Equilibrium↗
PubMed1990

Molecular cloning of a cDNA for rat hepatic glutaminase. Sequence similarity to kidney-type glutaminase.

Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase which plays an important role in the regulation of glutamine catabolism. Antibodies to hepatic glutaminase were used to screen a lambda gt11 rat liver cDNA library. One cDNA to hepatic glutaminase was identified. Changes in the relative abundance of hepatic glutaminase mRNA were determined by hybridization to this cDNA. The mRNA is found only in liver; it is not present prior to birth but its abundance increases dramatically at birth. The abundance of the mRNA is increased approximately 4-fold in diabetes. The sequence of the cDNA was compared to that recently published for kidney (brain)-type glutaminase (Banner, C., Hwang, J.-J., Shapiro, R.A., Wenthold, R.J., Nakatani, Y., Lampel, K.A., Thomas, J.W., Huie, D., and Curthoys, N.P. (1988) Mol. Brain Res. 3, 247-254). When the predicted amino acid sequences were compared a region of 123 amino acids with greater than 80% identity was found. The presence of scattered amino acid substitutions within stretches of identical amino acids suggests that the glutaminase isozymes are encoded by separate genes. This is the first demonstration of any similarity between the two glutaminases at the molecular level.

Aging↗
PubMed1990

Distribution of hepatic glutaminase activity and mRNA in perivenous and periportal rat hepatocytes.

Perivenous and periportal hepatocytes were isolated by the digitonin/collagenase perfusion technique. The specific activity of phosphate-activated glutaminase was 2.33-fold higher in periportal cells than in perivenous cells. Similarly, the relative abundance of glutaminase mRNA was 2.6-fold higher in samples from periportal cells. The distribution of glutaminase activity and mRNA was compared with those for glutamine synthetase (predominantly perivenous) and phosphoenolpyruvate carboxykinase (predominantly periportal). The results suggest that phosphate-activated glutaminase is predominantly expressed in the periportal zone of the liver acinus.

Animals↗
PubMed1990

Differential regulation of hepatic carbonic anhydrase isozymes in the streptozotocin-diabetic rat.

Most work with the male rat liver carbonic anhydrase isozymes in the past decade has centered on the cytosolic CA III and the mitochondrial CA V. This paper reports that the relative activity of both isozymes is altered in streptozotocin-diabetes. Carbonic anhydrase activity of perfused liver homogenates and disrupted, isolated mitochondria was measured by the mass spectrometric 18O decay technique at 37 degrees C. The contributions of the different isozymes were determined based on intracellular location and sensitivity to acetazolamide inhibition. Diabetes resulted in a twofold increase in the activity of CA V but a halving in the activity of CA III. This is the first time that liver CA V has been shown to be altered by physiological stress. The total carbonic anhydrase activity in the diabetic rat liver was unaltered compared with control rats; however, CA III never accounted for more than 50% of this activity. Since CA isozymes I, II, and IV together account for 30% of the CA activity in control rats and 70% in diabetic rats it is concluded that one or more of these isozymes is subject to regulation in the diabetic male rat. The increase in CA V during diabetes is in accord with this isozyme having an important function in provision of substrate for hepatic gluconeogenesis and ureagenesis.

Acetazolamide↗
PubMed1989

Effects of metabolic acidosis and diabetes on the abundance of specific renal mRNAs.

1. The effects of exogenously (NH4Cl ingestion) and endogenously (streptozotocin-diabetes) generated chronic metabolic acidosis on the abundance of rat renal mRNAs have been examined. 2. Total RNA was translated in vitro and the translation products analyzed by two-dimensional gel electrophoresis. 3. The translation product identified as phosphoenolpyruvate carboxykinase (PEPCK) increased 3.5-fold in both acidosis and diabetes. 4. This increase was not observed in diabetic rats treated with NaHCO3. 5. The abundance of one other translation product increased in acidosis. 6. That of 10 others increased in diabetes, several of which were elevated regardless of acid-base status. 7. The abundance of one translation product decreased in acidosis and diabetes but not in NaHCo3 treated diabetic rats, indicating acid-base regulation of this product. 8. The results establish that the acidosis response is limited to a small number of renal mRNAs and confirm that renal PEPCK is primarily regulated by changes in acid-base status. 9. They also indicate that diabetes affects the abundance of specific renal mRNAs through mechanisms independent of acid-base status.

Acidosis↗
PubMed1989

Phosphoenolpyruvate carboxykinase of rat small intestine: distribution and regulation of activity and mRNA levels.

Phosphoenolpyruvate carboxykinase (PEPCK) activity is present along the length of rat small intestine and in enterocytes throughout the villus-crypt axis. There is no detectable activity in submucosal layers. Messenger RNA encoding PEPCK is detectable in rat intestinal mucosa and the relative abundance increases markedly (3- to 8-fold) during starvation or streptozotocin-diabetes. However, these changes are not matched by changes in enzyme activity which are only slightly increased (1.5-fold). The intestine of neonatal rats possesses relatively high amounts of both PEPCK activity and mRNA. Based on the distribution and regulation of intestinal PEPCK, it is proposed that the enzyme does not play a significant role in either gluconeogenesis or glutamine catabolism in adult rats.

Animals↗
PubMed1989

Hormonal and nutritional regulation of phosphoenolpyruvate carboxykinase mRNA levels in chicken kidney.

In chickens, the kidney possesses a distinct cytosolic phosphoenolpyruvate carboxykinase activity which is not found in the liver. This activity is subject to long-term regulation by diet and changes in acid-base status. The activity is increased during starvation or metabolic acidosis. In addition, an unidentified component of some standard chicken diets results in altered activity. Using a specific cDNA probe the abundance of PEPCK mRNA has been determined in chicken kidney in vivo and in vitro. The abundance of PEPCK mRNA in chicken kidney increases during starvation and is rapidly decreased after refeeding carbohydrate. In isolated kidney tubules the abundance of the mRNA is increased after incubation with glucocorticoids, dibutyryl cAMP or hormones acting via changes in the concentration of cAMP (parathyroid hormone, epinephrine). Phorbol esters or hormones acting via calcium-dependent mechanisms were without effect. The results support the hypothesis that in the chicken the kidney is the major site of gluconeogenesis from substrates other than lactate and thus plays an important role in the maintenance of glucose homeostasis.

Animal Nutritional Physiological Phenomena↗
PubMed1988

Rat hepatic glutaminase: purification and immunochemical characterization.

A method for the purification of phosphate-activated glutaminase from the liver of streptozotocin-diabetic rats is described. The procedure involves solubilization of glutaminase activity from isolated mitochondria by sonication, followed by ammonium sulfate precipitation, polyethylene glycol precipitation, and sequential chromatography on DEAE, hydroxylapatite, and zinc-chelated resins. The enzyme was purified 600-fold to a specific activity of 31-57 U/mg protein. The purified enzyme has an apparent subunit molecular mass of 58,000-Da and is greater than 80% pure by scanning densitometry of sodium dodecyl sulfate-polyacrylamide gels. The purified enzyme has an apparent Km for glutamine of 17 mM and a pH optimum between 7.8 and 8.2. The physical and kinetic properties of this enzyme are similar to those of the enzyme from normal rat liver. Polyclonal antibodies raised against the enzyme specifically inhibit hepatic glutaminase activity and react primarily with a 58,000-Da peptide in liver fractions on immunoblots. These antibodies were used in equivalence point titrations and immunoblots to provide evidence for increased concentration of glutaminase protein in the liver of diabetic rats with no change in specific activity of the enzyme. In addition, the antibodies cross-react, at low affinity, with kidney-type glutaminases. On immunoblots, the antibodies did not react with fetal liver, mammary gland, or lung. Antibodies to rat hepatic glutaminase should prove useful as tools to study the long-term regulation of the enzyme.

Ammonium Sulfate↗
PubMed1987

The regulation of glutamine and ketone-body metabolism in the small intestine of the long-term (40-day) streptozotocin-diabetic rat.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.

Animals↗