Yu-Yan Yeh

Ketone bodies serve as important precursors of brain lipids in the developing rat.

Ketone bodies are readily oxidized for energy by extrahepatic tissues. Since oxidation of ketone bodies produces acetyl coenzyme A (AcCoA), and hence could be an important source of immediate precursors for fatty acid synthesis, we investigated, in whole-brain homogenates of developing rats, the preferential utilization of [3-14C]acetoacetate (AcAc), [3-14C]β-hydroxybutyrate (β-OHB), and [U-14C]glucose for production of CO2 and lipids, including phospholipids, glycerides, cholesterol, and free fatty acids. Throughout the postnatal period, the rate of AcAc oxidation was 2–3 and 2–6 times the rate for β-OHB and glucose, respectively. The eynthesis of lipids from AcAc was 7- to 11-fold higher than from glucose. The brain’s capacity for lipid synthesis from β-OHB was similar to that from AcAc during the first 8 days of life; however, during the next 10 days, the synthesis of lipids from β-OHB decreased to 60% of AcAc-dependent synthesis. The high rate of lipid synthesis from ketone bodies was accompanied by increased activities of cytoplasmic acetoacetyl CoA synthetase and acetoacetyl CoA thiolase in the developing brain. During the entire postnatal development, the proportion of radioactivity claimed by lipids vs. CO2 from [3-14C]AcAc was 44–62% vs. 38–56%; from [3-14C]β-OHB, 50–81% vs. 19–50%; and from [U-14C]glucose, 14–43% vs. 57–86%. Phospholipids accounted for more than two-thirds of total lipids synthesized from either ketone bodies or glucose, while diglycerides plus cholesterol and free fatty acids accounted for most of the remainder. Addition of glucose to the incubation medium did not alter lipid production from AcAc throughout the suckling period, but moderately depressed energy production in the brain of 16- to 20-day-old rats. It is clear that in cell-free preparations from the brain of developing rats, ketone bodies are preferred over glucose as precursors for both energy and lipids, mainly phospholipids. These results suggest that ketone bodies are important for the growth and development of the brain.

Relative utilization of fatty acids for synthesis of ketone bodies and complex lipids in the liver of developing rats.

The regulation of hepatic ketogenesis, as related to the metabolism of fatty acids through oxidative and synthetic pathways, was studied in developing rats. [1-14C] palmitate was used as a substrate to determine the proportions of free fatty acids utilized for the production of ketone bodies, CO2 and complex lipids. Similar developmental patterns of hepatic ketogenesis were obtained by measuring the production of either [14C]acetoacetate from exogenous [1-14C] palmitate or the sum of unlabeled acetoacetate and β-hydroxybutyrate from endogenous fatty acids. The production of total ketone bodies was low during the late fetal stage and at birth, but increased rapidly to a maximum value within 24 hr after birth. The maximal ketogenic capacity appeared to be maintained for the first 10 days of life.14CO2 production from [1-14C] palmitate increased by two- to fourfold during the suckling period, from its initial low rate seen at birth. The capacity for synthesis of total complex lipids was low at birth and had increased by day 3 to a maximal value, which was comparable to that of adult fed rats. The high lipogenic capacity lasted throughout the remaining suckling period. When ketogenesis was inhibited by 4-pentenoic acid, the rate of synthesis of complex lipids did not increase despite an increase in unutilized fatty acids. During the mid-suckling period, approximately equal amounts of [1-14C] palmitate were utilized for the synthesis of ketone plus CO2 and for complex lipid synthesis. By contrast, in adult fed rats, the incorporation of fatty acids into complex lipids was four times higher than that of ketone plus CO2. These observations suggest that stimulated hepatic ketogenesis in suckling rats results from the rapid oxidation of fatty acids and consequent increased production of acetyl CoA, but not from impaired capacity for synthsis of complex lipids.

Insulin, A Possible Regulator of Ketosis in Newborn and Suckling Rats

Extract: A possible regulatory role of insulin in the development of ketosis in newborn and suckling rats was inrestigated. The average plasma concentration of total ketone bodies measured at birth was 0.414 ± 0.037 μmol/ml. Within 24 hr after birth the level of ketones had increased to 4 times its initial value. The 3-to 4-fold increase in plasma ketones was maintained during the first 5 days of life but started to decline thereafter. Plasma insulin of newborn rats at birth (62 ± 8 μU/ml) was comparable to that of fed adult rats (85 ± 10 μU/ml). The levels decreased to 28 μU/ml on the first day of life and stayed low throughout the suckling period despite a tendency to increase at the time close to weaning. The capacities for ketone production in liver homogenates of suckling rats were inversely related to the levels of insulin. Administration of insulin (0.125 mU/g body weight, im) and glucose (1.75 mg/g body weight, ip) both suppressed plasma ketone bodies in suckling rats. Insulin administration increased plasma insulin but failed to decrease plasma glucose. Injection of glucose increased plasma insulin and glucose. Neither insulin nor glucose treatment changed the plasma levels of free fatty acids. These data suggest that a limited availability of insulin permits a high rat of ketogenesis and hence induced ketosis in newborn and suckling rats.Speculation: Developing rats suckled by their dams derive most of their energy from the high fat and low carbohydrate content of milk. The low concentration of insulin in suckling rats not only minimizes utilization of glucose by insulin-dependent tissues but permits a rapid synthesis of ketone bodies that then serve as energy sources for extrahepatic tissues, particularly the brain. Consequently, the energy requirements of suckling rats can be met with a reduced risk of hypoglycemia. Further studies on the effect of insulin on lipolysis, fatty acid oxidation, and ketone synthesis in vitro could add to our understanding of the action of insulin in reversing ketosis of suckling rats.

Partition of ketone bodies into cholesterol and fatty acids in vivo in different brain regions of developing rats

The proportions of labeled ketone bodies and glucose incorporated into cholesterol and fatty acids in different regions of the brain in developing rats were compared. In cerebrums of 15- and 18-day-old rats, the ratios of dpm cholesterol/dpm fatty acids incorporated from [3-14C] acetoacetate and [3-14C] β-hydroxybutyrate ranged from 0.4 to 0.7, or 50 to 100% higher than values obtained with [U-14C] glucose. Much higher ratios were obtained with younger animals: from 1 to 12 days of life, the values ranged from 1.0 to 1.3 with [3-14C] β-hydroxybutyrate as substrate, and, from 1 to 5 days, with [3-14C] acetoacetate, they were 1.0 or greater. During the first 12 days of life, the ratios resulting from administration of [U-14C] glucose were 0.4–0.7. Clearly, a greater proportion of acetoacetate and β-hydroxybutyrate was incorporated into cholesterol during the first week of life than the remaining suckling period. Like cerebrum, other brain regions (i.e., cerebellum, midbrain, brain stem and thalamus) yielded higher ratios of dpm cholesterol/dpm fatty acids from [3-14C] β-hydroxybutyrate during the first 12 days of life than on day 17. Brain stem was the most active region for lipid synthesis, and had the highest dpm cholesterol/dpm fatty acid ratio. Since active synthesis of cholesterol from ketone bodies during the early postnatal period coincides with a period of rapid brain growth, the results indicate that ketone bodies are more important early in the suckling period as sources of cholesterol for brain growth.

Pulmonary surfactant lipid synthesis from ketone bodies, lactate and glucose in newborn rats

The contribution of acetoacetate (AcAc), β-hydroxybutyrate (βOHB), lactate and glucose to pulmonary surfactant lipid synthesis in three-to five-day-old rats was measured. Minced lung tissue was incubated with3H2O and [3-14C]AcAc, [3-14C]βOHB, [U-14C]lactate or [U-14C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60–80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14–20%. In the presence of unlabeled glucose,14C-labeled AcAc, lactate and βOHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of βOHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of β-hydroxybutyrate dehydrogenase measured for newborn lung. These results demonstrate that AcAc and lactate are important precursors for surfactant lipids in neonatal rat lung.

Fatty acid oxidation in isolated rat liver mitochondria: Developmental changes and their relation to hepatic levels of carnitine and glycogen and to carnitine acyltransferase activity☆

Abstract Prompted by an apparent relationship between ketosis and fatty acid utilization, we studied the capacities for fatty acid oxidation through β-oxidation and Krebs cycle in liver mitochondria isolated from fetal and suckling rats. Rates of state 3 oxidation, as measured by oxygen consumption, were low for both palmitylcarnitine and palmityl CoA plus carnitine at 2 days before term and at birth, but increased at least ninefold during the first 8 days of life and at least sixfold during the remaining suckling period. Despite these sharp increases, oxygen consumption in suckling rats did not exceed the value for fed adult rats. Also, the rates of state 3 oxidation of succinate were low in suckling rats. Respiratory control indices, determined with each of the three substrates, were lower in suckling rats than fed adults. By contrast, ratios of fatty acyl ester to succinate oxidation, a relative measure of the oxidation of palmitylcarnitine and palmityl CoA, were 21–66% and 27–77% higher in suckling than in fed adult rats. The increased ratios indicate that the capacity for fatty acid oxidation is higher during postnatal development than in the fetal stage or adulthood. The oxidation capacity was inversely related to glycogen content in the liver. Although hepatic carnitine concentration and carnitine palmityltransferase activity increased during suckling period, they are not rate limiting for fatty acid oxidation. Studies of the partitioning of fatty acids showed that about two-thirds of the fatty acid oxidized through β-oxidation did not enter Krebs cycle for further oxidation. These results support our working hypothesis that ketosis of suckling rats stems from rapid oxidation of fatty acids and increased partitioning of acetyl CoA into ketogenesis.

Lung lipid synthesis from acetoacetate and glucose in developing rats in vitro

Acetoacetate (AcAc) and glucose were compared as energy sources and as precursors for lipid synthesis in the lungs of developing rats. Minced lung tissue was incubated with [3-14C]AcAc or [U-14C]glucose and the oxidation of each substrate to CO2 or its incorporation into tissue lipids was quantified. The highest rates of oxidation were obtained during the first 5 days for AcAc and the first 2 days of life for glucose and oxidation of AcAc was 3–4 times greater than that of glucose at all ages. Throughout postnatal development, the rates of nonsaponifiable lipid, fatty acid and hence total lipid (chloroform/methanol extractable) synthesis from AcAc were 2–3 times those of glucose. The highest rates of total lipid synthesis from AcAc and glucose were observed at birth. Glucose was utilized for glyceride-glycerol synthesis at a higher rate than AcAc. Similar patterns of incorporation of AcAc and glucose into various lipid classes were noted. Of the total lipids synthesized from AcAc and glucose, respectively, phospholipid plus monoglyceride accounted for 64% and 77%, triglyceride 13% and 13%, diglyceride plus cholesterol 11% and 4% fatty acids 9% and 4%, and cholesteryl esters 3% and 1%. At birth, the specific activities of all lipids except triglyceride derived from AcAc were greater than those from glucose. Rates of synthesis of all complex lipids declined with age. The results of these experiments demonstrate that AcAc is utilized more readily than glucose for energy production and lipid synthesis in developing rat lungs.

Pathways of acetyl CoA production for lipogenesis from acetoacetate, β-hydroxybutyrate, pyruvate and glucose in neonatal rat lung

The rate of fatty acid synthesis from acetoacetate (AcAc) is 2–3 times greater than from glucose in developing rat lung. To determine the reason for this difference, we investigated the pathways of lipogenesis from [3-14C] AcAc, [3-14C]β-hydroxybutyrate (βOHB), [U-14C] glucose or [2-14C] pyruvate in minced lung tissue of 3- to 4-day-old rats. The addition of (−)hydroxycitrate, an inhibitor of ATP-citrate lyase, inhibited fatty acid synthesis from glucose, pyruvate, and βOHB by 88%, 70% and 60%, respectively, but had no effect on that from AcAc. Benzene 1,2,3-tricarboxylate, an inhibitor of tricarboxylate translocase, inhibited fatty acid synthesis from all substrates by at least 50%. Incubation with aminooxyacetate, an inhibitor of aspartate aminotransferase, has no effect on lipid synthesis from glucose, pyruvate or AcAc, but increased lipid synthesis from βOHB. Results indicate that for lipid synthesis in the neonatal lung, acetyl CoA from AcAc is derived predominantly from a cytoplasmic pathway involving AcAcCoA synthetase and AcAcCoA thiolase, whereas citrate is the major route of acetyl group transfer from glucose. Lipogenesis from βOHB involves both the cytoplasmic and citrate pathways.

Ketone body synthesis from leucine by adipose tissue from different sites in the rat

Leucine is catabolized to ketone bodies in adipose tissue, but the contribution of this output to overall ketone metabolism is not known. The intent of the present study was to determine the capacity of different adipose tissues to synthesize ketone bodies from leucine. The amino acid was readily converted into acetoacetate in epididymal, perirenal, and omental fat tissues. In rats fed ad libitum, the rate of acetoacetate synthesis in omental fat (about 2 mumol g tissue-1h-1) was at least 8 times higher than in epididymal or perirenal fat. In omental fat, the rates of acetoacetate formation from alpha-ketoisocaproic acid were 47-55% lower than from leucine at all concentrations examined. There was no significant synthesis of beta-hydroxybutyrate from leucine or alpha-ketoisocaproic acid. After oxidative decarboxylation, a greater proportion (about three-fourths) of leucine in omental fat was metabolized to acetoacetate than to CO2 production through the Krebs cycle. Although addition of glucose, pyruvate, or carnitine did not affect the production of acetoacetate, fasting for 24 h stimulated acetoacetate synthesis from leucine and alpha-ketoisocaproic acid in omental fat. The high rate of leucine conversion to acetoacetate in omental fat was related to high activities of leucine aminotransferase and branched-chain alpha-keto acid dehydrogenase. Moreover, protein content and cytochrome c oxidase activity of omental mitochondria were, respectively, 13 and 12 times higher than in epididymal mitochondria. In contrast, fat content of epididymal adipose tissue was 21 times that of omental adipose tissue. Epididymal depot consisted of 2.0% protein and 75.8% fat, whereas omental depot contains 17.2% protein and 3.6% fat, resembling that of liver and muscle. The results suggest that the high ketogenic capacity of omental fat stems in part from an augmented mitochondrial mass and high activity of branched-chain alpha-keto acid dehydrogenase.