George Dunaway

A review of animal phosphofructokinase isozymes with an emphasis on their physiological role

SummaryPhosphofructokinase (PFK) isozymes and their physiological significance have been the focus of extensive research. The majority of this work has been centered around the PFK isozymes of rat, human and rabbit tissues. Consequently, this review emphasizes these studies. Additionally, a review of PFK isozymes in chickens, mice, guinea pig, and pig is presented. The relationship of the properties of each PFK isozyme in different tissues to the rates of glycolysis and/ or gluconeogenesis in those tissues is discussed where possible. Moreover, the contribution of the different PFK isozymes to alterations of the glycolytic rate in various tissues is discussed in relationship to variations in nutritional, hormonal, developmental or pathological status of the animal.

An animal model of gestational diabetes

Glucose tolerance tests (GTTs), hemoglobin A, levels, and pup weights were studied in both normal (C57BL/KsJ-+m/ + m) and heterozygous (C57BL/KsJ-db+/ + m) mice. There was no difference in GTT or hemoglobin A, levels between heterozygotes and normal animals in the nonpregnant state. However, the pregnant heterozygous mouse had significantly elevated GTTs (p less than 0.001) and hemoglobin A, levels (p less than 0.002) when compared to the normal pregnant mouse. The mean weight of pups from heterozygous parents was significantly greater than that of pups from normal parents (p less than 0.0005). Because the heterozygous mouse (C57BL/KsJ-db+/ + m) exhibits these characteristics of gestational diabetes, it may be possible to use it as an animal model of gestational diabetes.

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)

Characterization of the rat heart 6-phosphofructo-1-kinase isozymes

By elution with a discontinuous gradient from QAE-Sephadex, the rat muscle 6-phosphofructo-1-kinase (PFK) isozyme (M4) and the major rat liver PFK isozyme (L4) could be completely separated. Subjecting heart supernatant fluid to this treatment indicated that all of the heart PFK activity was found in the first wash, where M4 eluted, indicating little, if any, L4 was present. However, about 50% of the heart PFK activity was immunoprecipitated by L4 anti-IgG demonstrating the presence of the L-type subunit. A purification procedure was developed which yielded an enzyme preparation of high specific activity and resulted in a recovery of 50% to 60% of the original PFK activity. Three proteins were detected in this PFK preparation that exhibited apparent mol wt of 87 500, 85 000 and 80 000. The 85 000 and 80 000 Dalton components corresponded to the subunits of M4 and L4, respectively. The third protein was thought to be a distinct subunit (C-type) since it exhibited the highest molecular weight and was present in an exhaustively washed immunoprecipitate of purified heart PFK. From seven different heart PFK preparations, the relative distributions of the L-, M-, and C-type subunits were 0.80 +/- 0.1, 4.5 +/- 0.7, and 0.7 +/- 0.1, respectively. A comparison of the kinetic properties of L4, M4, and purified heart PFK isozymes clearly demonstrated that all three preparations exhibited different regulatory properties. In ventricular and atrial preparations the total PFK activity and the relative amounts of each subunit were drastically different suggesting regional differences between the distributions of PFK isozymes and consequently in regulation of PFK activities and glycolysis.

Purification of homogeneous rat phosphofructokinase isozymes with high specific activities

The purification of rat muscle and liver phosphofructokinase (PFK) isozymes has been greatly facilitated by column chromatographic separation on immobilized Cibacron Blue F3GA. The homogeneous liver PFK isozyme exhibited a specific activity of greater than 200 units per mg of protein which is nearly two-fold greater than has been previously reported for this isozyme. The yields for this isozyme exceeded 40% of the original activity and the molecular weight of its subunit was about 85,000 as determined by SDS-polyacrylamide gel electrophoresis. The muscle PFK isozymes specific activity was approximately 265 units/mg of protein which also is about twice the greatest specific activity previously reported. The overall yield for muscle PFK exceeded 50% of the original activity, and the molecular weight of its subunit was approximately 82,000. Using each homogeneous isozyme, antibodies were produced in rabbits; and the immunoglobin-G (IgG) fraction from the sera of these rabbits was highly specific for the PFK isozyme used as an antigen.

Regulation of skeletal muscle 6-phosphofructo-1-kinase during aging and development.

The purpose of this paper is to provide insight into the alterations of 6-phosphofructo-1-kinase (PFK) activity and isozyme types of rat skeletal muscle during development and aging. PFK isozymes are tetramers which may be comprised of one or any combination of the three subunit types, L, M, and C. The effects of fusion or terminal differentiation of cultured rat L6 myoblasts leading to formation of myotubles does not have a noticeable effect on total PFK activity. However, the amount of M-type subunit was increased; and the level of the C-type subunit decreased. These subunit changes caused shifts in the isozymic types. The ultimate effects of prenatal development of PFK were characterized in the near-term fetal muscle. This stage of development was accompanied by a significant loss of the C-type subunit and by two-fold increases in the L-type and M-type subunits which accounted for the 40% increase in total PFK activity. After birth, the M-type subunit increased dramatically as did the total PFK activity. Since the L-type and C-type subunits were gradually lost during the subsequent 3 weeks, the homotetramer of the M-type subunit (M4) was the only type which is present in mature muscle. M4 persisted as the only detectable form in skeletal muscle during the remainder of life, but the total PFK activity and amount of M4 decreased after 18 months of age. The decreased total PFK activity in aged skeletal muscle suggested that the expression of PFK genes may have reverted to an immature state when total PFK activity was lower. As shown by both the immunological analysis and direct quantification of subunit types, this clearly did not occur. That is, the loss of PFK activity in aged muscle is a consequence of decreased levels of the M-type subunit and not reappearance of other subunit types such as found in maturing muscle. Further, our examination of aged skeletal muscle indicates that no significant structural changes in M-type subunits had occurred and that inactive or partially active proteins which could crossreact with the M-type subunit were not detectable. It is suggested that the loss of M4 could cause a depression of the glycolytic rate leading to diminished ability of senile muscle to accommodate extreme energy demands.

Gender and post-ischemic recovery of hypertrophied rat hearts.

BackgroundGender influences the cardiac response to prolonged increases in workload, with differences at structural, functional, and molecular levels. However, it is unknown if post-ischemic function or metabolism of female hypertrophied hearts differ from male hypertrophied hearts. Thus, we tested the hypothesis that gender influences post-ischemic function of pressure-overload hypertrophied hearts and determined if the effect of gender on post-ischemic outcome could be explained by differences in metabolism, especially the catabolic fate of glucose.MethodsFunction and metabolism of isolated working hearts from sham-operated and aortic-constricted male and female Sprague-Dawley rats before and after 20 min of no-flow ischemia (N = 17 to 27 per group) were compared. Parallel series of hearts were perfused with Krebs-Henseleit solution containing 5.5 mM [5-3H/U-14C]-glucose, 1.2 mM [1-14C]-palmitate, 0.5 mM [U-14C]-lactate, and 100 mU/L insulin to measure glycolysis and glucose oxidation in one series and oxidation of palmitate and lactate in the second. Statistical analysis was performed using two-way analysis of variance. The sequential rejective Bonferroni procedure was used to correct for multiple comparisons and tests.ResultsFemale gender negatively influenced post-ischemic function of non-hypertrophied hearts, but did not significantly influence function of hypertrophied hearts after ischemia such that mass-corrected hypertrophied heart function did not differ between genders. Before ischemia, glycolysis was accelerated in hypertrophied hearts, but to a greater extent in males, and did not differ between male and female non-hypertrophied hearts. Glycolysis fell in all groups after ischemia, except in non-hypertrophied female hearts, with the reduction in glycolysis after ischemia being greatest in males. Post-ischemic glycolytic rates were, therefore, similarly accelerated in hypertrophied male and female hearts and higher in female than male non-hypertrophied hearts. Glucose oxidation was lower in female than male hearts and was unaffected by hypertrophy or ischemia. Consequently, non-oxidative catabolism of glucose after ischemia was lowest in male non-hypertrophied hearts and comparably elevated in hypertrophied hearts of both sexes. These differences in non-oxidative glucose catabolism were inversely related to post-ischemic functional recovery.ConclusionGender does not significantly influence post-ischemic function of hypertrophied hearts, even though female sex is detrimental to post-ischemic function in non-hypertrophied hearts. Differences in glucose catabolism may contribute to hypertrophy-induced and gender-related differences in post-ischemic function.

Distribution of hepatic phosphofructokinase isozymes in parenchymal and sinusoidal cells

Abstract The distribution profile of the isozymes of phosphofructokinase (PFK) in different cell types of rat liver is established using the techniques of electrophoresis and immunodiffusion. Agarose gel electrophoresis of the extracts of parenchymal cells, Kupffer or sinusoidal cells, and whole liver indicated that two PFK isozymes are present in whole liver and that the faster moving hepatic PFK isozyme is present only in parenchymal cells; whereas, the slower moving hepatic PFK isozyme is only in sinusoidal cells. Immunodiffusion studies using antiserum specific for the major hepatic PFK isozyme (PFK-L2) revealed that PFK-L2 is present only in whole liver or parenchymal cell extracts and is absent from sinusoidal cells. It is apparent that the other hepatic PFK isozyme (PFK-L1) is normally found only in sinusoidal cells.

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-l-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 (7507o). 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.

Teaching pharmacology within a multidisciplinary organ system-based medical curriculum.

Abstract. We have described how the pharmacology of agents that act on the central nervous system (CNS) and endocrine system were incorporated into a case-based, multidisciplinary, integrated sophomore medical curriculum at Southern Illinois University School of Medicine (SIUSM). Faculty members from the Departments of Pharmacology, Pathology, Internal Medicine, Psychiatry, and Neurology were major participants in the CNS block, and faculty with primary expertise in radiology, epidemiology, and immunology also participated. Integrated sessions involving the entire class were organized around brief patient cases, which were given to the students in advance of the session. During the sessions, each patient case was presented by a physician or pathologist who briefly described symptoms, history, physical findings, differential diagnosis, and the classification of the subtypes of the particular class of disorder. The pathophysiology of the disease was discussed, and the pharmacology of agents to be considered for therapy was presented in the context of the Pharmacology Mental Algorithm, a systematic and rational approach to drug therapy. The session was completed by a clinician who added further clinically relevant information, which was followed by a question-and-answer period involving all faculty participants. The CNS block was presented over a 1-month period and included standardized patients and real patients who consistently exhibited specific disease findings, and these patient-oriented sessions were followed with small group tutorial sessions. Single discipline large-group sessions were also used to present material that is introductory or unique to a particular discipline. Student knowledge was assessed, using integrated evaluations based on case vignettes with multiple-choice questions provided by each discipline. The goal of the examination was to evaluate knowledge base in the discipline areas and its application to clinical problem-solving. A practical evaluation of each students patient examination skills in CNS was also performed by clinicians. Feedback from the students on the organ system activities was obtained using a questionnaire. Development of these sessions required leadership and a considerable amount of time to organize, improve, and update sessions. The integrated approach largely eliminated the use of conflicting terminology and redundancy of material common with separate presentations on the same subject by different disciplines. The multidisciplinary, case-based sessions were perceived to be instructive, valuable learning experiences, based on formal and informal student and faculty feedback.