Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes.

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].     

Hormonal regulation of ketogenesis in hepatocytes from fed rats before and after glycogen depletion

Vasopressin, angiotensin II and the catecholamines decreased ketogenesis from oleate but increased ketogenesis from butyrate in hepatocytes from fed rats. The hormones increase CO2 production from both oleate and butyrate. It is suggested that whereas the mitochondrial uptake of butyrate is linked to its rate of oxidation, that of oleate is independent of its intramitochondrial metabolism, and consequently the oxidation of oleate to CO2 occurs at the expense of ketogenesis. Effects of the hormones on ketogenesis from oleate or butyrate were not observed after pre-treatment of the hepatocytes with dibutyryl cyclic AMP for 1 hour. The insensitivity of ketogenesis to the hormones after this treatment (which mimics the effects of acute carbohydrate deprivation in vivo) questions the physiological significance of hormonal regulation of ketogenesis other than at the onset of starvation.     

Rates of ketone-body formation in the perfused rat liver

1. The rates of formation of acetoacetate and β-hydroxybutyrate by the isolated perfused rat liver were measured under various conditions. 2. The rates found after addition of butyrate, octanoate, oleate and linoleate were about 100μmoles/hr./g. wet wt. in the liver of starved rats. These rates are much higher than those found with rat liver slices. 3. The differences between the rates given by slices and by the perfused organ were much higher with the long-chain than with short-chain fatty acids. The increments caused by oleate and linoleate were 12 and 16 times as large in the perfused organ as in the slices, whereas the increments caused by butyrate and octanoate were about four times as large. 4. The rates of ketogenesis in the unsupplemented perfused liver of well-fed rats, and the increments caused by the addition of fatty acids, were about half of those in the liver from starved rats. 5. The value of the [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was raised by octanoate, oleate and linoleate. 6. Carnitine did not significantly accelerate ketogenesis from fatty acids. 7. Oleate formed up to 82% of the expected yield of ketone bodies. 8. In the liver of alloxan-diabetic rats the endogenous rates of ketogenesis were raised, in some cases as high as in the liver from starved rats, after addition of oleate. 9. On addition of either β-hydroxybutyrate or acetoacetate to the perfusion medium the liver gradually adjusted the [β-hydroxybutyrate]/[acetoacetate] ratio towards the normal range. 10. The [β-hydroxybutyrate]/[acetoacetate] ratio of the medium was about 0·4 when slices were incubated, but near the physiological value of 2 when the liver was perfused. 11. The experiments demonstrate that for the study of ketogenesis slices are in many ways grossly inferior to the perfused liver.     

Evidence for a reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed virgin and lactating rats.

Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1--14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265--270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529--2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.     

Requirements of glycerol and fatty acid for triglyceride synthesis and ketogenesis by hepatocytes from normal and triiodothyronine-treated rats

Hepatocytes from T3-treated rats synthesized less triglyceride and more ketone bodies from [1-14C]oleate at all concentrations from 0-2 mM, than did hepatocytes from euthyroid animals; addition of 1.0 mM glycerol increased triglyceride synthesis and reduced ketogenesis in hepatocytes from T3-treated rats to the rates observed in euthyroid hepatocytes in the absence of added glycerol. Glycerol did not alter triglyceride synthesis, but reduced ketogenesis genesis by euthyroid hepatocytes. It is probable from these and other data (J. Biol. Chem. 259, 8857-8862 (1985)) that, in the hyperthyroid rat, glycero-3-P, and not fatty acid, is rate limiting for synthesis of triglyceride, and, secondarily for reducing rates of ketogenesis in the hepatocyte.     

Adaptation of hepatic gluconeogenesis and ketogenesis to altered supply of substrates during late pregnancy in the rat

The relative importance of the main glucogenic and ketogenic substrates, and interactions between fatty acids availability and ketogenesis have been investigated in virgin or 21 day pregnant rats. Fed pregnant rats displayed elevated lactatemia and the production of lactate by portal-drained viscera was markedly reduced. In contrast, the production of alanine and propionate from digestion was almost similar in fed pregnant and virgin rats. The release of glucose by the liver in fed animals was higher in pregnant rats, and lactate was the main glucogenic substrate taken up whereas alanine uptake was reduced. The hepatic utilization of propionate was not different between the two groups of fed animals. Hepatic gluconeogenesis and lactate extraction were enhanced by starvation; the contribution of lactate to glucose release remained higher in pregnant than in virgin rats, whereas the contribution of alanine was lower, owing to its decreased availability in afferent blood. There was a large uptake of intestinally-derived acetate in fed rates, and a slight release, parallel to ketogenesis, was observed in starved pregnant rats. Free fatty acids were elevated and efficiently taken up by the liver in fed pregnant rats, but without any noticeable ketogenesis. Hepatic ketogenesis was enhanced in starved animals, with marked hyperketonaemia in pregnant rats. However, in those animals, the hepatic release of ketone bodies was not proportional to ketonaemia and was almost similar to the release in starved virgin rats.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Quantitation of ketogenesis in periportal and pericentral regions of the liver lobule

A method has been devised to quantitate rates of ketogenesis (acetoacetate + beta-hydroxybutyrate production) in discrete regions of the liver lobule based on changes in NADH fluorescence. In perfused livers from fasted rats, ketogenesis was inhibited nearly completely with either 2-bromoctanoate (600 microM) or 2-tetradecylglycidic acid (25 microM). During inhibition of ketogenesis, a linear relationship (r = 0.90) was observed between decreases in NADH fluorescence detected from the liver surface and decreases in ketone body production. NADH fluorescence was monitored subsequently from individual regions of the liver lobule by placing microlight guides on periportal and pericentral regions of the liver lobule visible on the liver surface. Rates of ketogenesis in sublobular regions were calculated from regional decreases in NADH fluorescence and changes in the rate of ketone body formation by the whole liver during infusion of inhibitors. In the presence of bromoctanoate, ketogenesis was reduced 80% and local rates of ketogenesis were decreased 31 +/- 4 mumol/g/h in periportal areas and 28 +/- 3 mumol/g/h in pericentral regions. Similar results were observed with tetradecylglycidic acid. Therefore, it was concluded that submaximal rates of ketogenesis from endogenous, mainly long-chain fatty acids are nearly equal in periportal and pericentral regions of the liver lobule in liver from fasted rats. Rates of ketogenesis and NADH fluorescence were strongly correlated during fatty acid infusion. Infusion of 250 microM oleate increased NADH fluorescence maximally by 8 +/- 1% over basal values in periportal regions and 17 +/- 4% in pericentral areas. Local rates of ketogenesis, calculated from these changes in fluorescence, increased 35 +/- 6 mumol/g/h in periportal areas and 55 +/- 5 mumol/g/h in pericentral regions. Thus, oleate stimulated ketogenesis nearly 60% more in pericentral than in periportal regions of the liver lobule.     

Effect of lactation on gluconeogenesis and ketogenesis in ovine hepatocytes

1. Rates of glucose synthesis from radioactive precursors and ketogenesis were determined in hepatocytes from control and lactating sheep. 2. Gluconeogenesis from propionate was the same in both groups. Gluconeogenesis from lactate + pyruvate was three-fold higher in hepatocytes from lactating sheep. Palmitate stimulated gluconeogenesis from lactate + pyruvate in both groups. 3. Rates of ketogenesis from palmitate but not butyrate were slightly higher in hepatocytes from lactating sheep. No other differences in the metabolism of palmitate or butyrate were seen in the two groups. Exogenous carnitine stimulated ketogenesis from palmitate. Propionate inhibited ketogenesis from palmitate and butyrate. Lactate + pyruvate also inhibited ketogenesis slightly but stimulated oxidation and esterification. 4. It is concluded that the major changes in glucose and ketone production seen in the lactating ruminant are not the result of long-term changes within the hepatocyte but occur because of the changes in substrate supply to the liver and changes in intracellular concentrations of metabolites.     

Conversion of pyruvate into ketone bodies in rat hepatocyte suspensions.

The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.     

Physiological aspects of the regulation of ketogenesis

The importance of ketone bodies (acetoacetate and 3-hydroxybutyrate) as substrates for peripheral tissues, especially nervous tissue, of man is now firmly established. This has renewed interest in the factors that control the production of ketone bodies by the liver in various physiological situations, such as alterations of dietary status, stage of development or alteration in demand for circulating substrates (e.g. in exercise or lactation). In the discussion of the regulation of ketogenesis in the present paper, distinction is made between extrahepatic and intrahepatic control. The former is mainly concerned with the factors (e.g. hormonal status of animals) that alter the flux of non-esterified fatty acids to the liver, whereas intrahepatic regulation involves the fate (esterification versus beta-oxidation) of fatty acids within the liver. Emphasis is placed on the fact that alterations in blood glucose concentrations are indirectly responsible, via effects on insulin secretion, for the extrahepatic control of ketogenesis. By analogy, it is postulated that the carbohydrate status of the liver may play a role in the intrahepatic regulation of ketogenesis. Some support for this postulate is provided by comparison of measurements of blood ketone-body concentrations in various inborn errors of hepatic carbohydrate metabolism (e.g. deficiencies of glucose 6-phosphatase, fructose 1,6-bisphosphatase and glycogen synthase) in man and by experiments with isolated rat hepatocytes. Present information on the short- and long-term factors that may be responsible for the altered rates of ketogenesis during the foetal-neonatal and suckling-weanling transitions, in lactation, on feeding a high-fat diet and post-exercise is discussed. It is concluded that the major factors involved in the regulation of ketogenesis in these situations are (a) flux of non-esterified fatty acids to the liver and (b) the partitioning of long-chain acyl-CoA between the esterification and beta-oxidation pathways.     

Interactions between vasopressin and glucagon on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats.

Vasopressin (10nM) inhibited ketogenesis (56%) in hepatocytes from fed rats when oleate (1 mM) was the substrate, but had no effect with butyrate (10mM). The hormone increased the accumulation of lactate and stimulated the esterification of [1(-14)C]oleate (70%). These effects of vasopressin were reversed by glucagon (10 nM). The physiological implications of these findings are discussed.     

The fuel of respiration of rat kidney cortex

1. In kidney-cortex slices from the well-fed rat, glucose (5mm) supplied 25–30% of the respiratory fuel; in the starved state, the corresponding value was 10%. These results are based on measurements of the net uptake of glucose and of the specific radioactivity of labelled carbon dioxide formed in the presence of [U-¹⁴C]-glucose. 2. Added acetoacetate (5mm) or butyrate (10mm) provided up to 80%, and added oleate (2mm) up to 50% of the fuel of respiration. The oxidation of endogenous substrates was suppressed correspondingly. 3. More [U-¹⁴C]oleate was removed by the tissue than could be oxidized by the amount of oxygen taken up; less than 25% of the oleate removed was converted into respiratory carbon dioxide and about two-thirds was incorporated into the tissue lipids. The rate of oleate incorporation into the neutral-lipid fraction was calculated to be equivalent to the rate of oxidation of endogenous fat, which provided the chief remaining fuel. 4. The contribution of endogenous substrates to the respiration (50%) in the presence of added oleate is taken to reflect either a high turnover rate of the endogenous neutral lipids (approx. half-life 2·5hr.) or a raised rate of lipolysis caused by the experimental conditions in vitro. 5. Added l-α-glycerophosphate (2·5mm) increased oleate incorporation into the neutral-lipid fraction by up to 40% (i.e. caused a net synthesis of triglyceride). 6. Lactate (2·5mm) added as sole substrate supplied 30% of the respiratory fuel, but with added oleate (2mm) lactate was converted quantitatively into glucose. Oleate stimulated the rate of gluconeogenesis from lactate by 45%. 7. The oxidation of both long-chain and short-chain even-numbered fatty acids was accompanied by ketone-body formation. Ketone-body synthesis from oleate, but not from butyrate, increased six- to seven-fold after 48hr. of starvation. The maximum rates of renal ketogenesis (80μmoles/hr./g. dry wt., with butyrate) were about 20% of the maximum rates observed in the liver (on a weight-for-weight basis) and accounted for, at most, 35% of the fatty acid removed. 8. dl-Carnitine (1·0mm) had no effect on the rates of uptake of acetate, butyrate or oleate or on the rate of radioactive carbon dioxide formation from [U-¹⁴C]oleate, but increased ketone-body formation from oleate by more than 100%. Ketone-body formation from butyrate was not increased. 9. There is evidence supporting the assumption that there are cells in which gluconeogenesis and ketogenesis occur together, characterized by equal labelling of [U-¹⁴C]oleate and the ketone bodies formed, and other cells that oxidize fat and do not form ketone bodies. 10. Inhibitory effects of unlabelled acetoacetate on the oxidation of [1-¹⁴C]butyrate and of unlabelled butyrate on [4-¹⁴C]acetoacetate oxidation show that fatty acids and ketone bodies compete as fuels on the basis of their relative concentrations. 11. The pathway of ketogenesis in renal cortex must differ from that of the liver, as β-hydroxy-β-methylglutaryl-CoA synthetase is virtually absent from the kidney. In contrast with the liver the kidney possesses 3-oxo acid CoA-transferase (EC 2.8.3.5), and the ready reversibility of this reaction and that of thiolase (EC 2.3.1.9) provide a mechanism for ketone-body formation from acetyl-CoA. This mechanism may apply to extrahepatic tissues generally, with the possible exception of the epithelium of the rumen and intestines.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Metabolic fate of non-esterified fatty acids in isolated hepatocytes from newborn and young pigs. Evidence for a limited capacity for oxidation and increased capacity for esterification.

In hepatocytes isolated from 48 h-old starved of suckling newborn pigs or from 15-day-old starved piglets, the rate of ketogenesis from oleate or from octanoate is very low. This is not due to an inappropriate fatty acid uptake by the isolated liver cells, but results from a limited capacity for fatty acid oxidation. Some 80-95% of oleate taken up is converted into esterified fats, whatever the age or the nutritional conditions. Three lines of indirect evidences suggest that fatty acid oxidation is not controlled primarily by malonyl-CoA concentration in newborn pig liver. Firstly, the addition of glucagon does not increase fatty acid oxidation or ketogenesis. Secondly, the rate of lipogenesis is very low in isolated hepatocytes from newborn pigs. Thirdly, the rates of oxidation and ketogenesis from octanoate are also decreased in isolated hepatocytes from newborn and young piglets. The huge rate of esterification of fatty acids in the liver of the newborn pigs probably represents a species-specific difference in intrahepatic fatty acid metabolism.     

Effects of norepinephrine on the metabolism of fatty acids with different chain lengths in the perfused rat liver

The effects of norepinephrine on ketogenesis in isolated hepatocytes have been reported as ranging from stimulation to inhibition. The present work was planned with the aim of clarifying these discrepancies. The experimental system was the once-through perfused liver from fasted and fed rats. Fatty acids with chain lengths varying from 8-18 were infused. The effects of norepinephrine depended on the metabolic state of the rat and on the nature of the fatty acid. Norepinephrine clearly inhibited ketogenesis from long-chain fatty acids (stearate > palmitate > oleate), but had little effect on ketogenesis from medium-chain fatty acids (octanoate and laureate). With palmitate the decrease in oxygen uptake was restricted to the substrate stimulated portion; with stearate, the decrease exceeded the substrate stimulated portion; with oleate, oxygen uptake was transiently inhibited. Withdrawal of Ca²⁺ attenuated the inhibitory effects. ¹⁴CO₂ production from [1-¹⁴C]oleate was inhibited. Net uptake of the fatty acids was not affected by norepinephrine. In livers from fed rats, oxygen uptake and ketogenesis from stearate were only transiently inhibited. The conclusions are: (a) in the fasted state norepinephrine reduces ketogenesis and respiration by means of a Ca²⁺-dependent mechanism; (b) the degree of inhibition varies with the chain length and the degree of saturation of the fatty acids; (c) norepinephrine favours esterification of the activated long-chain fatty acids in detriment to oxidation; (d) in the fed state the stimulatory action of norepinephrine on glycogen catabolism induces conditions which are able to reverse inhibition of ketogenesis and oxygen uptake.     

Metabolism of octanoyl- and palmitoylcarnitine by intact rat hepatocytes

Ketogenesis from extracellular substrates was quantified using intact rat hepatocytes. Rates of ketogenesis from octanoylcarnitine and palmitoylcarnitine were 20 and 30%, respectively, of the rates observed from the corresponding free acids. In contrast, the free acids and the acylcarnitines were converted to ketone bodies at similar rates in a liver homogenate system. These results suggest that medium- and long-chain acylcarnitines may be relatively poor substrates for metabolism by intact liver cells.     

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.     

Development and regulation of ketogenesis in hepatocytes isolated from newborn rats.

The development of fatty acid metabolism was studied in isolated hepatocytes from newborn rats. Ketone-body production from oleate is increased 6-fold between 0 and 16 h after birth. This increase is related to an enhanced beta-oxidation rather than to a channeling of acetyl-CoA from the tricarboxylic acid cycle to ketone-body synthesis. The increase in oleate oxidation is not related to a decreased esterification rate, as the latter is already low at birth and does not decrease further. At birth, lipogenic rate is 2-3-fold lower than in fed adult rats and it decreases to undetectable values in 16 h-old rats. A 90% inhibition of lipogenesis in hepatocytes of newborn rats (0 h) by glucagon and 5-(tetradecyloxy)-2-furoic acid does not lead to an increased oxidation of non-esterified fatty acids. This suggests that the inverse relationship between lipogenesis and ketogenesis in the starved newborn rat is not responsible for the switch-on of fatty acid oxidation at birth. Moreover, ketogenesis from octanoate, a medium-chain fatty acid the oxidation of which is independent of carnitine acyltransferase, follows the same developmental pattern at birth as that from oleate.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.