Kenneth R. Swiatek

Lactate, 3-hydroxybutyrate, and glucose as substrates for the early postnatal rat brain

The dependence of cerebral energy metabolism upon glucose, 3-hydroxybutyrate, and lactate as fuel sources during the postnatal period was investigated. The brain of 6 day old suckling pups used very little glucose, but by the 15th postnatal day glucose was the major catabolite. Hydroxybutyrate was not a major brain fuel at either 6 or 15 days of age. Its utilization accounted for only 19% of the brains total energy needs at 15 days of age, even though blood ketone concentrations are near maximal at this time. Seventy percent of the cerebral metabolic requirements were met by lactate in animals aged 6 days. The major role played by lactate as a substrate for brain metabolism in young pups was not a result of abnormally elevated blood lactate concentrations. The slow catabolism of glucose in young brain can not be explained by low rates of influx or inadequate enzymatic capacity.

Hypoglycemia in the Newborn

There is no universal definition for hypoglycemia. Various investigators have empirically recommended different blood lucose levels (BGLs) that should be maintained in neonatal period to prevent injury to the developing brain. The “normal” range of blood glucose is variable and depends upon factors like birth-weight, gestational age, body stores, feeding status, availability of energy sources as well as the presence or absence of disease. Further, there is no concrete evidence to show the causation of adverse long-term outcomes by a particular level or duration of hypoglycemia. Hence, a consensus has been to evolve an “operational threshold”.

The inefficient transfer of maternally fed alcohol to nursing rats

The growth of rat pups nursed by ethanol-drinking mothers or pups exposed daily to ethanol/vapor was monitored for the first three weeks of life. Pups nursed by mothers fed either a 6% ethanol containing liquid diet or a 10% ethanol/water mixture had significantly lower body weights after the 2nd postnatal week than controls. This decrease in pup growth occurred despite pup blood alcohol levels that did not exceed 20 mg%. Only a small fraction of the alcohol fed to mothers ever reached suckling animals. In contrast, pups exposed daily to ethanol vapor regularly achieved blood alcohol concentrations in excess of 250 mg%, but experienced only minimal growth retardation. These results suggest that maternal alcohol feeding cannot be used to study the effects of ethanol upon postnatal development.

The metabolism of d- and l-3-hydroxybutyrate in developing rat brain

The incorporation of L- and D-3-hydroxybutyrate into rat brain protein, lipid, and amino acids during development was studied. L-3-Hydroxybutyrate was found to label brain protein and amino acids in addition to sterols and fatty acids throughout the first 32 postnatal days. Age related changes in L- and D-3-hydroxybutyrate labeling of protein and amino acids were similar. Whereas L-3-hydroxybutyrate incorporation into brain lipids rose sharply between 6-15 days of age, D-3-HOB incorporation into the lipid fraction gradually increased from birth through the age of 15 days. Incorporation by both isomers into lipid was greatest during the third week of suckling and then declined when the animals were weaned. At 15 days of age, the distribution of L-3-hydroxybutyrate into glutamate, glutamine + aspartate, and gamma-aminobutyrate was similar to that obtained with D-3-hydroxybutyrate. L-3-Hydroxybutyrate was poorly oxidized to CO2 by brain slices and mitochondria. Oxidation capacity was maximal from 15-21 days of age for both isomers. The activity of L-3-hydroxybutyrl-CoA ligase increased between 6-28 days of age, and its increase is well correlated with the developmental pattern of L-3-hydroxybutyrate incorporation and mitochondrial oxidation. L-3-Hydroxybutyrate was not detected in the blood of palmitate-injected pups or fasted adult animals. These results suggest that although L-3-hydroxybutyrate can be utilized for the synthesis of brain components during development, its negligible blood concentration precludes a significant contribution to either tissue synthesis or energy balance during the suckling period.

Enzymatic adaptations in newborn pig liver

Abstract Numerous studies during the neonatal period in the rat have demonstrated that many changes occur in the activities of liver enzymes of carbohydrate metabolism especially those of glycolysis, gluconeogenesis, and glycogen synthesis and degradation. Such information, however, has not been available in the newborn pig. The present study of several regulatory enzymes was undertaken to determine if an enzymatic defect could explain fasting hypoglycemia in the newborn pig. The hepatic liver enzymes, non-specific hexokinase, glucokinase, glucose-6-phosphatase, UDPG: α-glucan glucosyltransferase, phosphorylase, phosphofructokinase, aldolase, fructose diphosphatase, and phosphopyruvate carboxylase have been measured in developing pig liver. The activity of all hepatic enzymes except hexokinase were low in fetal tissue and increased after birth in livers from both fed and fasted pigs. Hexokinase activity was present prior to birth and remained essentially unchanged after birth and was not affected by prolonged starvation. Liver glycogen was rapidly mobilized at birth in both fed and fasted newborn animals. It is concluded that a defect in enzymes measured under the stated assay conditions does not exist and can not be offered as an explanation for the observed fasting hypoglycemia in newborn pigs.

Development of gluconeogenesis in pig liver slices

Abstract • Several studies have shown that newborn pigs are unable to maintain normal blood sugar when fasted immediately after birth. Among the reasons postulated were a defect or late maturation in a key regulatory enzyme of gluconeogenesis. Recently, glucose-6-phosphate, fructose diphosphate and phosphopyruvate carboxylase activities were shown to be low in fetal tissue and to increase within 48 h to levels observed in older normal glycemic animals. The present study was undertaken to determine if an enzymatic defect existed in pyruvate carboxylase and to determine the rates of gluconeogenesis in pig liver with respect to development and maturation. • The hepatic enzyme pyruvate carboxylase was found to be low in 1-day-old pigs and to increase 3–4 fold within 4 days. In addition, fructokinase was found to be completely absent in newborn liver and to remain low in animals until 10 days of age. The conversion rate of fructose to glucose in liver slices was found to follow the enzymatic developmental pattern. • The conversion of precursor substrates such as lactate, pyruvate, glycerol and dihydroxyacetone to glucose did not occur in 1-day-old, 24-h-fasted piglets. A significant increase in hepatic gluconeogenesis occurred 2–6 days postnatally and a further increase was found at 10–16 days of life. • The present study demonstrates that the newborn pig liver has a decreased gluconeogenic capacity in comparison to older pig liver and that the low rate may help to explain why the neonatal pig has difficulty in maintaining normal blood glucose when fasted immediately after birth. The relatively low rate of lactate and pyruvate conversion to glucose for 1–3 days may possible be enzyme limited although both pyruvate carboxylase and phosphopyruvate carboxylase are elevated by the 3rd day of life to levels found in older normal glycemic fasted pigs.

Distribution of phosphopyruvate carboxylase in pig liver

Abstract Phosphopyruvate carboxylase (GTP:oxaloacetate carboxylase (transphorylating), EC 4.1.1.32) activity has been found in pig and lamb liver mitochondria and in the soluble cell fraction in rat, hamster, and mouse livers. In pig liver, the cellular distribution was found to be 60–70% soluble and 15–25% mitochondrial. This cellular distribution was similar in both fed and 72-h fasted pigs. The specific activity increased 2 fold in the mitochondria and 3 fold in the soluble fraction prior to sacrifice. The K m constants for pig phosphopyruvate carboxylase at pH 6.4 obtained from the 100 000 × g cell supernatant were phosphoenolpyruvate, 6.65·10−4 M; Mn2+, 9.33·10−4 M; NaHCO3, 1.47·10−2 M; and IDP, 5.4·10−5 M.

Metabolism of l- and d-3-hydroxybutyrate by rat liver during development

Abstract The metabolism of d - and l -3-hydroxybutyrate by neonatal and suckling rats was investigated. Both isomers of 3-hydroxybutyrate were incorporated into hepatic lipid, amino acids, and protein throughout the developmental period. The enzyme activities of liver 3-oxo-CoA transferase and brain 3-hydroxybutyrate dehydrogenase were compared during the first 3 postnatal weeks. The results suggest that the enzymatic activity of liver 3-oxo-CoA transferase is sufficient to account for a major portion of the d -isomer incorporation. The production of CO 2 by rat liver was greater from d -3-hydroxybutyrate than that measured from the l -isomer. The in vitro oxidation of both the d - and l -isomers by rat liver was stimulated by ATP + CoA or by GTP + CoA which suggests that their utilization may also be mediated by acyl-CoA synthetase enzymes.

The utilization of dl-[3-14c]hydroxybutyrate by malnourished rat pups

Abstract Protein-calorie restricted newborn rats were found to incorporate significantly less β-hydroxybutyrate into brain protein and lipid components for the first 15 days of life as compared to well-fed controls. The labelling of brain tissue by β-hydroxybutyrate was maximal during mid-suckling in both groups and then decreased immediately after weaning to the levels measured in adult rats. Undernutrition did not alter the incorporation of β-hydroxybutyrate into brain amino acids. Unexpectedly, liver tissue was found capable of incorporating β-hydroxybutyrate into the protein, amino acids and lipid fractions to nearly the same extent as brain tissue although its utilization increased with age in contrast to the results observed for brain tissue. Malnutrition decreased the incorporation of β-hydroxybutyrate into liver protein but did not alter its entry into the amino acids or lipid components. The brain or liver enzymes, β-hydroxybutyrate dehydrogenase, 3-oxo-CoA transferase, or acetoacetyl coenzyme A thiolase activities were not affected by the dietary restriction. Differences in these enzymes therefore cannot explain the decreased incorporation of β-hydroxybutyrate into the brain or liver fractions. It is suggested that the underutilization of β-hydroxybutyrate by malnourished animals for protein and lipid formation is not due to decreased uptake, but may be due to an alteration in the protein and lipid synthetic capacities of the tissues. Since the amount of radioactive β-hydroxybutyrate entering the liver tissue fractions increased throughout development and hepatic 3-oxo-coenzyme A transferase activity is of the same order of magnitude as the brain β-ydroxybutyrate dehydrogenase, the transferase activity may after all be sufficient to account for the labelling observed in liver.

Evidence for the existence of enzymatic variants of β-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria

Abstract A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at −20°. The brain dehydrogenase was found to have a larger K m (apparent) for the 3-acetylpyridine analog of NAD + , and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD + and β-hydroxybutyrate. The K m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.