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Biomedical subjects

M Watford

Publications and source records attributed to M Watford.

At least 19 recordsLinked to original sources
PubMed2005

Antihyperglycemic activity of Tarralin, an ethanolic extract of Artemisia dracunculus L.

The studies reported here were undertaken to examine the antihyperglycemic activity of an ethanolic extract of Artemisia dracunculus L., called Tarralin in diabetic and non-diabetic animals. In genetically diabetic KK-A(gamma) mice, Tarralin treatment by gavage (500 mg/kg body wt./day for 7 days) lowered elevated blood glucose levels by 24% from 479+/-25 to 352+/-16 mg/dl relative to control animals. In comparison, treatment with the known antidiabetic drugs, troglitazone (30 mg/kg body wt./day) and metformin (300 mg/kg body wt./day), decreased blood glucose concentrations by 28% and 41%, respectively. Blood insulin concentrations were reduced in the KK-A(gamma) mice by 33% with Tarralin, 48% with troglitazone and 52% with metformin. In (STZ)-induced diabetic mice, Tarralin treatment, (500 mg/kg body wt./day for 7 days), also significantly lowered blood glucose concentrations, by 20%, from 429+/-41 to 376+/-58 mg/dl relative to control. As a possible mechanism, Tarralin was shown to significantly decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression by 28% in STZ-induced diabetic rats. In non-diabetic animals, treatment with Tarralin did not significantly alter PEPCK expression, blood glucose or insulin concentrations. The extract was also shown to increase the binding of glucagon-like peptide (GLP-1) to its receptor in vitro. These results indicate that Tarralin has antihyperglycemic activity and a potential role in the management of diabetic states.

Animals↗
PubMed2000

Dietary glutamine suppresses endogenous glutamine turnover in the rat.

Plasma glutamine turnover was determined using 1-14C-labeled glutamine in rats that consumed crystalline amino acid diets containing the equivalent of 16% protein with 25% of the amino acids as glutamine or a control diet containing no glutamine (or glutamate) for 10 days. Glutamine turnover in glutamine-fed animals was 66% of the rate in the control group. Glutamine feeding caused 20% higher levels of arterial plasma glutamine. Arterial-portal differences across the portal-drained viscera showed net glutamine uptake in control animals but no net uptake or release in the glutamine-fed group. Skeletal muscle glutamine synthetase activity was similar in both groups. The results indicate that long-term consumption of relatively large amounts of dietary glutamine decreases the turnover of plasma glutamine and thus reduces the need for endogenous glutamine synthesis.

Amino Acids↗
PubMed2000

Glutamine and glutamate metabolism across the liver sinusoid.

The liver shows net glutamine uptake after a protein-containing meal, during uncontrolled diabetes, sepsis and short-term starvation, but changes to net release during long-term starvation and metabolic acidosis. Some studies report a small net release of glutamate by the liver. The differential expression of glutamine synthetase (perivenous) and glutaminase (periportal) within the liver indicates that glutamine is used for urea synthesis in periportal cells, whereas glutamine synthesis serves to detoxify any residual ammonia in perivenous cells. Experiments in vivo suggest that changes in net hepatic glutamine balance are due predominantly to regulation of glutaminase activity, with the flux through glutamine synthetase being relatively constant.

Ammonia↗
PubMed2000

A cortisol surge mediates the enhanced expression of pig intestinal pyrroline-5-carboxylate synthase during weaning.

Citrulline synthesis from glutamine is enhanced remarkably in enterocytes of weanling pigs, but the molecular mechanism(s) involved are not known. The objective of this study was to determine whether a cortisol surge mediates the enhanced expression of intestinal citrulline-synthetic enzymes during weaning. Jejunal enterocytes were prepared from 29-d-old weanling pigs treated with or without metyrapone (an inhibitor of cortisol synthesis), or from age-matched unweaned pigs. The mRNA levels and activities of phosphate-dependent glutaminase (PDG), pyrroline-5-carboxylate synthase (P5CS), ornithine aminotransferase (OAT), carbamoyl-phosphate synthase I (CPS-I) and ornithine carbamoyltransferase (OCT) were determined. The mRNA levels for PDG, P5CS, OAT and OCT were 139, 157, 102 and 55% higher, respectively, in weanling pigs compared with suckling pigs. The activities of PDG and P5CS were 38 and 692% higher, respectively, in weanling pigs compared with unweaned pigs, but the activities of OAT, CPS-I and OCT did not differ between these two groups of pigs. The effects of metyrapone administration to weanling pigs were as follows: 1) prevention of a cortisol surge, 2) abolition of the increases in both mRNA levels and activity of P5CS, 3) no alteration in the mRNA levels and activities of PDG and CPS-I, 4) increases in the mRNA levels for OAT (216%) and OCT (39%) and in OAT activity (30%), and 5) prevention of the increase in intestinal synthesis of citrulline from glutamine. These results suggest that increased P5CS activity reflects in large part the increased levels of P5CS mRNA and is responsible for the increased synthesis of citrulline from glutamine in enterocytes of weanling pigs; these increases may be mediated by a cortisol surge during weaning that can be blocked by metyrapone administration.

Animals↗
PubMed2000

Functional glycerol kinase activity and the possibility of a major role for glyceroneogenesis in mammalian skeletal muscle.

According to textbook descriptions of glycerol metabolism, liver and kidney are the only tissues that express significant glycerol kinase activity. Thus esterification of fatty acids to triglycerides in peripheral tissues such as skeletal muscle and adipose tissue is presumed to be dependent on the synthesis of glycerol-3-phosphate from glucose. This report describes exciting new data indicating that, although low, the glycerol kinase activity of skeletal muscle is functional. Interestingly, the results also suggest that neither glycerol nor glucose is the major substrate for the synthesis of muscle triglyceride glycerol. Rather, glyceroneogenesis, the synthesis of glycerol-3-phosphate from lactate, may play an as yet under-appreciated, but quantitatively important, role.

Animals↗
PubMed1998

Distribution of phosphate-activated glutaminase isozymes in the chicken: absence from liver but presence of high activity in pectoralis muscle.

The distribution of glutaminase expression in a uricotelic species, the chicken, has been examined using cDNA probes to the rat isozymes. The results suggest that chickens do not possess a glutaminase isozyme equivalent to the liver-type isozyme of mammalian liver. Measurements of enzymic activity also showed very low glutaminase activity in chicken liver. Extra-hepatic tissues in the chicken do express a glutaminase isozyme mRNA which is detected by rat kidney-type glutaminase cDNA. The abundance of this mRNA was highest in kidney and breast muscle and relatively abundant in brain, spleen and adipose tissue. Chicken small intestine expressed relatively low levels of the mRNA. The high level of glutaminase mRNA in chicken pectoralis muscle was accompanied by high glutaminase enzymic activity. In contrast, in mixed leg muscle glutaminase mRNA was barely detectable by Northern blot and glutaminase activity was relatively low. Starvation for 48 h resulted in a slight decrease in the activity of glutaminase in pectoralis muscle, but a large decrease in the relative abundance of the mRNA. The results suggest that in the chicken, hepatic glutamine hydrolysis is not quantitatively important, but skeletal muscle may be a major site of glutamine catabolism.

Animals↗
PubMed1997

Rat hepatic glutaminase: identification of the full coding sequence and characterization of a functional promoter.

Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5' untranslated sequence for rat hepatic glutaminase was isolated by screening lambda ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5' flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5' deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.

Amino Acid Sequence↗
PubMed1997

Rat adipose tissue amino acid metabolism in vivo as assessed by microdialysis and arteriovenous techniques.

In fed, anesthetized rats, microdialysis demonstrated a net release of glycerol, glutamine, serine, tyrosine, and taurine and a net uptake of glutamate, aspartate, glycine, and arginine across the inguinal adipose depot. However, the results also indicated excessive proteolysis associated with implantation of the microdialysis probe, and a novel arteriovenous difference technique was developed. Arteriovenous difference across the inguinal fat pat demonstrated a net uptake of glucose and a net release of lactate and glycerol. Starvation (48 h) resulted in higher rates of glycerol and lactate release with lower rates of glucose uptake. A net uptake of triacylglycerol was seen in starved-refed animals. Net glutamine, tyrosine, and taurine release was seen in fed and starved animals, but in starved-refed animals taurine and serine were the only amino acids showing significant release. No significant net uptake or release of ammonia, pyruvate, or alanine was observed. These experiments confirm that adipose tissue is a site of glutamine synthesis and suggest that the principal substrates are derived from intracellular proteolysis. The results also demonstrate the viability of an arteriovenous difference technique for the study of adipose tissue in the rat.

Adipocytes↗
PubMed1995

Regulation of glutaminase activity and glutamine metabolism.

Glutamine is synthesized primarily in skeletal muscle, lungs, and adipose tissue. Plasma glutamine plays an important role as a carrier of nitrogen, carbon, and energy between organs and is used for hepatic urea synthesis, for renal ammoniagenesis, for gluconeogenesis in both liver and kidney, and as a major respiratory fuel for many cells. The catabolism of glutamine is initiated by either of two isoforms of the mitochondrial glutaminase. Liver-type glutaminase is expressed only in periportal hepatocytes of the postnatal liver, where it effectively couples ammonia production with urea synthesis. Kidney-type glutaminase is abundant in kidney, brain, intestine, fetal liver, lymphocytes, and transformed cells, where the resulting ammonia is released without further metabolism. The two isoenzymes have different structural and kinetic properties that contribute to their function and short-term regulation. Although there is a high degree of identity in amino acid sequences, the two glutaminases are the products of different but related genes. The two isoenzymes are also subject to long-term regulation. Hepatic glutaminase is increased during starvation, diabetes, and feeding a high-protein diet, whereas kidney-type glutaminase is increased only in kidney in response to metabolic acidosis. The adaptations in hepatic glutaminase are mediated by changes in the rate of transcription, whereas kidney-type glutaminase is regulated at a posttranscriptional level.

Adipose Tissue↗
PubMed1994

Hepatic glutaminase mRNA is confined to part of the urea cycle domain in the adult rodent liver lobule.

This in situ hybridization study describes the developmental appearance of the lobular distribution of the mRNA encoding hepatic glutaminase in normal rat liver. Glutaminase has been proposed to provide the urea cycle with ammonia [Häussinger and Gerok (1983) Eur. J. Biochem. 133, 269-275]. Hence, the (developmental) pattern of expression of the mRNA would be expected to be closely linked to that of the urea cycle enzymes. From embryonic day 20 onward, hepatic glutaminase mRNA can be detected along the entire porto-central axis, with predominant expression in the portal area. In the adult phenotype, which is acquired at the end of the first postnatal week, glutaminase mRNA is no longer present along the entire porto-central distance but has become confined to a relatively small periportal domain in which the expression decreases in a porto-central direction. Thus, in contrast to the large periportal domain, in which the urea cycle enzymes are expressed, the glutaminase mRNA-expressing domain is much smaller and not contiguous with the glutamine synthase mRNA-expressing pericentral domain, leaving a midlobular area that is devoid of glutaminase mRNA. A similar pattern of distribution was found in adult mouse liver. The significance of these observations is that, within the liver lobules, there is an area in which glutaminase is not expressed and, hence, glutamine can not be the substrate for urea synthesis.

Animals↗
PubMed1994

Glutamine metabolism in rat small intestine: synthesis of three-carbon products in isolated enterocytes.

Glutamine is a major respiratory fuel for enterocytes but the extent of glutamine decarboxylation in these cells is not certain. The metabolism of differentially labeled L-[14C]glutamine was studied in enterocytes isolated from fed rats. The results indicate that glutamine undergoes two decarboxylations and yields a three carbon end product. The first decarboxylation is presumably at alpha-ketoglutarate dehydrogenase but the identity of the second reaction is not clear. The addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, was without effect on either the rate of glutamine metabolism or the extent of decarboxylation. Labeled glutamine carbon was recovered in three carbon products primarily as alanine with lesser amounts as lactate. The addition of glucose to the incubation medium did not change the rate of glutamine metabolism, or decarboxylation, but lactate became the major labeled three carbon end product. The results show that the fate, alanine or lactate, of glutamine derived pyruvate in enterocytes depends on the relative rate of flux through pyruvate and indicates that one cytosolic pool of pyruvate exists in these cells. The limited oxidation of glutamine in enterocytes ensures that the gluconeogenic potential of glutamine is conserved within the body.

Animals↗
PubMed1994

Transcriptional regulation of the hepatic glutaminase gene in the streptozotocin-diabetic rat.

1. Liver possesses a unique isozyme of phosphate activated glutaminase which is subject to long-term regulation. 2. In the rat streptozotocin-diabetes results in a 4-fold increase in the rate of transcription of the rat hepatic glutaminase gene. 3. This is consistent with previous reports from this laboratory of increases, of similar magnitude, in the relative abundance of hepatic glutaminase mRNA (Smith and Watford (1990) J. Biol. Chem. 265, 10631-10636), and enzyme activity (Watford, et al. (1984) Biochem. J. 224, 207-214). 4. The work establishes that, in contrast to the regulation of renal glutaminase where mRNA stability plays an important role, the predominant site of long-term regulation of hepatic glutaminase is at the level of gene transcription.

Animals↗
PubMed1994

Transcriptional control of rat hepatic glutaminase expression by dietary protein level and starvation.

Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase that is subject to long-term regulation. In rats during starvation or after consumption of diets containing high amounts of protein (60%), hepatic glutaminase activity was 100% higher than in rats fed a 20% protein diet. Conversely, rats fed low protein diets (0 and 5%) had lower hepatic glutaminase activity when compared with rats fed the 20% protein diet. Differences in activity with different dietary protein levels were not due to differences in the amount of food consumed. The relative abundance of mRNA encoding hepatic glutaminase was lower in rats fed 0% protein and higher in those starved or fed 60% protein diet when compared with rats fed the 20% protein diet. The mRNA elongation assay in hepatic nuclei isolated from these animals demonstrated that the rate of transcription of the glutaminase gene was also different in rats starved or fed different levels of dietary protein. Overall, the results indicate that differences in hepatic glutaminase activity in rats starved or fed different levels of protein are mainly due to differences in the rate of transcription of the gene. In this way the regulation of hepatic glutaminase expression is similar to that seen for other enzymes involved in hepatic amino acid catabolism but differs markedly from that of renal glutaminase, in which changes in transcription rate are not observed and alterations of mRNA turnover are the principle mechanism of long-term regulation.

Animals↗
PubMed1994

Production of glutamine and utilization of glutamate by rat subcutaneous adipose tissue in vivo.

Information about adipose tissue amino acid metabolism is limited, with most data derived from studies in vitro. The purpose of this study was to further characterize the role of adipose tissue in glutamine metabolism in the rat in vivo. The extracellular concentrations of glutamine, glutamate, alanine, and ammonia were measured in the rat inguinal fat pad using a microdialysis sampling technique. A calibration method was used to accurately assess the extracellular levels of metabolites, and a comparison of these concentrations with those in arterial blood allowed determination of the net flux of each compound. The adipose tissue-arterial blood concentration differences were 122 +/- 19, 54 +/- 37, -61 +/- 21, and -28 +/- 13 microM for glutamine, alanine, glutamate, and ammonia, respectively, indicating a production of glutamine and an uptake of glutamate by subcutaneous adipose tissue. The magnitude of glutamine production suggests that adipose tissue may play a significant role in whole body glutamine homeostasis.

Adipose Tissue↗
PubMed1993

Relationship between hepatic fatty acid oxidation and gluconeogenesis in the fasting neonatal pig.

Hepatocytes were isolated from sixteen fasting neonatal pigs and used in two experiments: (1) to determine the effect of various factors on the ability for hepatic oxidation of fatty acids and (2) to clarify the relationship between fatty acid oxidation and glucose synthesis. In Expt 1, newborn pigs were either fasted from birth for 24 h or allowed to suck ad lib. for 3 d followed by a 24 h fast. In the presence of pyruvate, oxidation of octanoate (2 mM) was about 30-fold greater than oleate (1 mM) regardless of age, but glucose synthesis was not enhanced beyond that observed for pyruvate alone. Inclusion of carnitine (1 mM), glucagon (100 nM) or dibutyryl cAMP (50 microM) in the incubation media did not stimulate either fatty acid oxidation (octanoate or oleate) or glucose synthesis. Extending the period of fasting to 48 h (Expt 2) failed to enhance the fatty acid oxidative capacity or glucose synthesis rate. Likewise, the redox potential of the gluconeogenic substrate (lactate v. pyruvate) did not influence glucose synthesis regardless of the oxidative capacity exhibited for fatty acids. These data indicate that fatty acid oxidative capacity is not the first limiting factor to full expression of gluconeogenesis in hepatocytes isolated from fasted newborn pigs.

Aging↗