Biomedical subjects

G A Dunaway

Publications and source records attributed to G A Dunaway.

At least 19 recordsLinked to original sources

PubMed1996

Alteration of 6-phosphofructo-1-kinase subunit protein, synthesis rates, and mRNA during rat neonatal development.

For the three 6-phosphofructo-1-kinase (PFK) subunits in heart, skeletal muscle, liver and kidney, developmentally-associated changes in protein, mRNA and apparent synthesis rates were observed. During neonatal maturation, all three phenomena for the M-type in heart and skeletal muscle exhibited large increases. Also, during neonatal development, the L-type and C-type subunits were unaffected in heart but disappeared from skeletal muscle. In the newborn liver and kidney, the amounts of each type of PFK subunit protein were nearly identical. During neonatal development, the levels of all three PFK subunit proteins in kidney increased more than twofold; and this was associated with a similar increase in apparent subunit synthesis rates and mRNA levels. During liver neonatal development, the L-type subunit protein, synthesis and mRNA levels also increased more than twofold. However, during hepatic maturation, M-type subunit protein, synthesis and mRNA levels were unchanged and apparently unaffected. The C-type subunit protein during neonatal liver development decreased approximately 80% as did its apparent synthesis rate. These data suggest that regulation of the alteration of the PFK subunit proteins during neonatal maturation can vary among these tissues and is not the same for each subunit type. Different mechanisms, such as transcription, translation, and mRNA stability could be involved.

Age Factors↗

PubMed1995

Alteration of PFK subunit protein, synthesis, and mRNA during neonatal brain development.

During neonatal maturation of rat brain, a similar biphasic relationship exists between the previously reported pattern of glucose utilization and levels of each type of 6-phosphofructo-1-kinase (PFK) subunit protein, relative synthesis, and mRNA. The increasing amounts of each subunit isoform generally correlated with elevated protein synthesis which was promoted by greater amounts of each type of subunit mRNA. For each parameter, the early phase, 1 to 10 days after birth, was characterized by small increases, and the subsequent period from ten to thirty days postpartum was characterized by a much greater rate of increase. By 30 days after birth, adult values were observed. The apparent efficiency of translation of each type of PFK subunit mRNA in brain suggests that the M-type subunit mRNA is the most efficient and that the L-type subunit mRNA is the least. The greatest relative increases in subunit protein, mRNA, and synthesis were observed for the C-type subunit. Since enhanced translation apparently makes little, if any, contribution, a possible explanation of these phenomena could be increased transcription of the PFK genes. These neonatal changes could involve age-dependent alteration of methylation of the PFK gene promotor(s) and/or activity of effectors of the transcription of the PFK genes.

Animals↗

PubMed1995

Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1).

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1993

Regulation of brain 6-phosphofructo-1-kinase: effects of aging, fructose-2,6-bisphosphate, and regional subunit distribution.

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P2 in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P2 in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P2 levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P2 due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.

Aging↗

PubMed1993

Fructose 2,6-bisphosphate: changes during neonatal maturation and aging of rat and potential role in regulation of glucose utilization.

During the 6 days following birth, tissue levels of fructose-2,6-P2 in rat brain, liver, muscle, heart and kidney did not significantly change. However, by the tenth day postpartum fructose-2,6-P2 levels in brain, heart, and skeletal muscle increased approximately 50% and attained adult values. During maturation of liver, adult levels of fructose-2,6-P2 were not achieved until 3-4 weeks after birth or approximately at the time of maximum rates of gluconeogenesis. Renal fructose-2,6-P2 levels in the neonate were initially elevated and 2-3 weeks after birth decreased approximately 2.5-fold to adult values. With the exception of the pons-medulla, which showed no significant changes in fructose-2,6-P2 amounts, levels of this regulatory sugar from aging brain regions were generally decreased. The fructose-2,6-P2 levels from heart atria of old rats (24-30 month) were also significantly decreased. In diaphragm, the fructose-2,6-P2 levels were increased at 12 months of age and at 27 months of age were twice the level at 3 months. The fructose-2,6-P2 levels during the aging of liver, skeletal muscle (EDL and soleus), spleen, thymus, kidney, testis and lung were not significantly altered.

Aging↗

PubMed1992

Characterization of phosphofructokinase-deficient canine erythrocytes.

Dogs homozygously affected with muscle-type phosphofructokinase (PFK) deficiency had about 20% of normal erythrocyte PFK activity and exhibited a compensated haemolytic anaemia. Erythrocyte glucose-6-phosphate and fructose-6-phosphate concentrations were increased and dihydroxyacetone phosphate and 2,3-bisphosphoglycerate values were below normal in affected dogs. Other intermediates distal to the PFK step were not significantly below normal and fructose-1,6-bisphosphate was even above normal. Erythrocyte ATP was higher than normal in affected dogs owing to the reticulocytes present. Abnormal adenylate metabolism was demonstrated by low ATP/AMP and ADP/AMP ratios and the inability to maintain ATP content when affected erythrocytes were incubated with cyanide. Glucose-1,6-bisphosphate content was normal, and fructose-2,6-bisphosphate content in affected canine erythrocytes was higher than normal. Studies of erythrocyte PFK isozymes revealed altered enzyme kinetic properties in affected dogs which appeared to be due to the loss of the M-type subunit.

Adenine Nucleotides↗

PubMed1992

Developmental changes of 6-phosphofructo-1-kinase subunit levels in erythrocytes from normal dogs and dogs affected by glycogen storage disease type VII.

1. The subunit proportions (L:M:C) of the PFK isozymes from normal adult erythrocytes were 2:86:12. Affected adult erythrocyte 6-phosphofructo-1-kinase (PFK) isozymes contained normal L-type (31%) and C-type (61%) subunits as well as a small amount (8%) of truncated M-type subunit. 2. When measured within 24 hr of birth, both normal and affected dog erythrocytes contained high PFK activities due to elevated levels of the L-type subunit. As the dogs matured, PFK activity decreased due to a greater than 99% loss of the L-type subunit. 3. By 2 weeks of age, the M-type and C-type subunits in normal dog PFK isozymes increased several-fold and attained near adult levels. 4. During post-natal development, the L-type subunit from affected dog erythrocytes decreased more rapidly than from normal dog erythrocytes; but it was maintained at a higher level in the affected adult erythrocytes. Also, in the affected dog erythrocytes, truncated M-type subunits were detected; and the initially high levels of the C-type subunit decreased approximately 50% after 4 weeks.

Aging↗

PubMed1991

The subunit proportions and kinetic properties of 6-phosphofructo-1-kinase isozymes from rat heart atria and ventricle progressively change during aging.

Relative to 2-3 month rats, total 6-phosphofructo-1-kinase (PFK) activity in heart atria from 12 month rats declined 31%; but, by 24 months it was decreased by only 13%. PFK activities from 12 and 24 month ventricles relative to the 2-3 month rats were decreased by 40% and 30%, respectively. This change in PFK activity in each heart region was associated with alterations of subunit composition. In heart atria from 12 and 24 month rats when compared to 3 month rats, the levels of L-type subunit were not significantly different; but the levels of the M-type subunit were decreased by 43% and 38%, respectively. With respect to levels in 2-3 month atria, the C-type subunit in 12 month atria decreased by 27%; and at 24 months it increased by 31%. Making the same comparison for the heart ventricle at 12 and 24 months, L-type subunit decreased by 30% and 24% respectively; M-type subunit decreased by approximately 47%; and the C-type subunit increased 1.9 and 4.7 fold, respectively. These age-related changes of subunit composition in atrial and ventricular PFK isozyme pools led to changes in their kinetic and regulatory properties suggesting that the aged rat could exhibit a diminished capacity to produce ATP from glucose.

Aging↗

PubMed1991

Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII.

6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31% of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.

Adenosine Triphosphate↗

PubMed1990

Age-related changes in subunit composition and regulation of hepatic 6-phosphofructo-1-kinase.

6-Phosphofructo-1-kinase (PFK) isoenzyme pools from livers of fetal, neonatal, young adult (3 months) and aged (24 months) rats were studied. Near-term liver PFK isoenzyme pools were composed of nearly equal quantities of all three subunits. During the 30 days after birth, the total activity increased by 25%; the amount of the L-type, M-type or C-type subunit was increased 3-fold, was unchanged, or was decreased by 80% respectively. In aged rats, compared with young adults, total PFK activity was unchanged, but the L-type, M-type or C-type subunit decreased by 24%, increased by 39%, or increased by 338% respectively. During neonatal maturation, the changing subunit composition of the hepatic isoenzyme pools led to a decreased susceptibility to ATP inhibition, to a greater apparent affinity for fructose 6-phosphate, and to increased sensitivity to fructose 2,6-bisphosphate. Also, these alterations correlated with the measured increases in fructose 2,6-bisphosphate and the reported optimal rate of hepatic glycolysis/gluconeogenesis.

Aging↗

PubMed1990

The effects of various anesthetics on tissue levels of fructose-2,6-bisphosphate in rats.

We report that the short-term use of various anesthetic agents prior to decapitation causes alteration of the levels of fructose-2,6-bisphosphate in kidney, brain, heart, muscle, and liver. These data indicate that even light anesthesia can not be used when levels of this metabolite are to be determined. Also, it appears that the use of any of these anesthetics can profoundly alter glucose utilization in many tissues.

Anesthetics↗

PubMed1989

Physiological relevance of the changing subunit composition and regulatory properties of the 6-phosphofructo-1-kinase isozyme pools during heart and muscle development.

During postnatal development, the subunit compositions of the 6-phosphofructo-1-kinase isozyme pools of heart and skeletal muscle are known to change. The isozyme pools from fetal muscle were composed of the L-type (60%), and M-type (36%) and C-type (4%) subunits and the isozymes from fetal and early neonatal heart contain nearly equal amounts of all three subunits. During postnatal development of both tissues, the proportion of the M-type subunit increases until it is the only type present in adult muscle and the major subunit in adult heart (75%). The isozyme pool from fetal muscle exhibit a decreased affinity for fructose-6-P and a greater susceptibility to ATP inhibition compared to the M-rich isozymes which are subsequently present. The isozyme pools from fetal and early neonatal heart, if compared to the M-rich isozymes which are present later during heart development and to the fetal muscle isozymes, exhibited the least affinity for fructose-6-P and the greatest susceptibility to ATP inhibition. Comparison of the isozyme pools containing little or no C-type subunit with those from fetal and early neonatal heart clearly indicates that the presence of substantial levels of the C-type subunit imposed a decreased ability for fructose-2,6-P2 to both lower affinity for fructose-6-P and antagonize sensitivity to ATP inhibition. Although still not thoroughly appreciated, it appears that the changing nature of the isozyme pools in these tissues permits regulation of glucose metabolism in a manner which allows efficient utilization of nutritional opportunities and which adequately meets the energy requirements of each tissue at different stages of development.

Adenosine Triphosphate↗

PubMed1988

Physiological implications of the alteration of 6-phosphofructo-1-kinase isozyme pools during brain development and aging.

The 6-phosphofructo-1-kinase (PFK) isozyme pools from brains of fetal, neonatal, young adult (3 months) and aged (30 months) rats were studied using chromatographic and immunological techniques. Also, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. The total PFK activity increased over seven-fold during the 30 days following birth, and the L-type, M-type, and C-type subunits increased approximately 2-fold, 7-fold, and 24-fold, respectively. In the near-term fetal brain and early neonatal brain, the L-type and M-type subunits were the predominant forms and were present in approximately equal amounts. During the second second week of postnatal brain maturation, the levels of the M-type and C-type subunit began to significantly increase. Consequently, during postnatal development, the isozyme pools switched from L-M-rich forms to M-C-rich forms. In aged brain relative to the young adult (3 months) brain, the 20% loss of total activity was associated with 27% and 18% losses of the M-type and C-type subunits, respectively. Examination of the regulatory properties of the various PFK isozyme pools revealed that at the low concentration of fructose-6-P and high level of ATP which are thought to occur in vivo, fructose-2,6-P2 was required for measurable PFK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1988

Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.

Adenosine Triphosphate↗

PubMed1987

Nature of the subunits of the 6-phosphofructo-1-kinase isoenzymes from rat tissues.

The nature of the PFK (6-phosphofructo-1-kinase) isoenzymes in many rat tissues was examined by immunological and chromatographic techniques and by measurement of their subunit compositions. It was revealed that, except for diaphragm and skeletal muscle, these complex isoenzymic populations contained different amounts of the three subunit types and were nearly tissue-specific. Apparently this tissue specificity is due to different concentrations of the tetramers, which in turn are controlled by the types and amounts of each subunit that are available to associate randomly.

Animals↗

PubMed1986

Alteration of 6-phosphofructo-1-kinase isozyme pools during heart development and aging.

The nature of 6-phosphofructo-1-kinase isozyme pools in fetal, neonatal, young adult (3 months), and aged (30 months) rat hearts was studied using chromatographic and immunological techniques. Furthermore, the changing subunit composition of each isozyme pool was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% slab gels and by immunoblotting with subunit-specific antibodies. Although all three subunit types were expressed in heart throughout life, total activity and the nature of the isozyme pools varied during neonatal development and in aged heart. In fetal heart, the complex tetramers containing all three subunits appeared to be the major isozyme types. As the heart matured to the young adult stage, the M-type subunit increased over 6-fold; whereas the changes in the other two subunits were considerably less. These data indicate that during neonatal heart maturation the isozymic pools progressively exhibited increased amounts of the tetrameric forms containing two or more M-type subunits. In aged heart relative to the young adult (3 months) heart, the total activity and proportion of M-type subunit in the isozymes were decreased; and consequently, the amounts of the M-rich isozymes were decreased. The shifts in the types of isozymes during heart maturation and subsequent aging were primarily due to changes in availability of the M-type subunit to participate in random assembly of the tetrameric isozymes.

Aging↗

PubMed1986

Are the rat tissue/organ proportions of 6-phosphofructo-1-kinase subunits strain-specific?

Recent studies suggest that the tissue/organ proportions of 6-phosphofructo-1-kinase (PFK) subunits from diverse strains of rat may be drastically different. To test this possibility rigorously, the PFK isoenzyme populations and subunit contents in muscle, liver, brain and heart were examined in the following strains: Wistar, ACI, Long Evans, Norway Brown and Wag/Rij. Regardless of the strain, adult muscle possessed only the M-type subunit; adult liver contained predominantly the L-type subunit as well as M-type and C-type subunits; and the adult brain and heart exhibited all three subunit types.

Animals↗

PubMed1986

Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase.

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.

Animals↗