Studies in the ketosis of fasting.

A series of experiments was performed during the induction of starvation ketosis and in the acute reversal of the ketotic state. In contrast to the predictions of two widely held theories of ketogenesis, control of acetoacetate production by the liver appeared to be unrelated to changes in fatty acid mobilization from the periphery, fatty acid oxidation, fatty acid synthesis, or the acetyl coenzyme A concentration in the liver.Ketosis of fasting was shown to be reversible within 5 minutes by the injection of glucose or insulin. This effect was due to a prompt cessation of acetoacetate production by the liver. The possibility is raised that the ketosis of fasting is due to a direct activation of acetoacetate-synthesizing enzymes secondary to a starvation-induced depression of insulin secretion by the pancreas.

Acute reversal of experimental diabetic ketoacidosis in the rat with (+)-decanoylcarnitine.

The effect of (+)-decanoylcarnitine, a potent inhibitor of long-chain acylcarnitine transferase, was tested for its ability to inhibit hepatic ketogenesis both in the isolated perfused liver and in vivo in severely ketotic alloxan diabetic rats. In vitro the inhibitor caused an almost complete block in ketone body production. In vivo (+)-decanoylcarnitine caused a rapid reversal of ketosis under conditions where large doses of insulin had little effect. A combination of the two agents produced an even more striking fall in plasma ketone levels.While (+)-decanoylcarnitine alone had no effect on plasma glucose levels it enhanced the hypoglycemic effect of insulin in anesthetized animals. Loss of this effect was noted in nonanesthetized animals, possibly as a result of increased muscle activity. Studies in the isolated perfused liver indicated that the blockade of fatty acid oxidation and ketogenesis produced by (+)-decanoylcarnitine was rapidly reversible upon removal of the inhibitor.

Hormonal control of ketogenesis. Rapid activation of hepatic ketogenic capacity in fed rats by anti-insulin serum and glucagon.

The enhanced capacity for long-chain fatty acid oxidation and ketogenesis that develops in the rat liver between 6 and 9 h after the onset of starvation was shown to be inducible much more rapidly by administration of anti-insulin serum or glucagon to fed rats. After only 1 h of treatment with either agent, the liver had clearly switched from a "nonketogenic" to a "ketogenic" profile, as determined by rates of acetoacetate and b-hydroxybutyrate production on perfusion with oleic acid. As was the case after starvation, the administration of insulin antibodies or glucagon resulted in depletion of hepatic glycogen stores and a proportional increase in the ability of the liver to oxidize long-chain fatty acids and (-)-octanoylcarnitine, suggesting that all three treatment schedules activated the carnitine acyltransferase system of enzymes. In contrast to anti-insulin serum, which produced marked elevations in plasma glucose, free fatty acid, and ketone body concentrations, glucagon treatment had little effect on any of these parameters, presumably due to enhanced insulin secretion after the initial stimulation of glycogenolysis. Thus, after treatment with glucagon alone, it was possible to obtain a "ketogenic" liver from a nonketotic animal. The results are consistent with the possibility that the activity of carnitine acyltransferase, and thus ketogenic capacity, is subject to bihormonal control through the relative blood concentrations of insulin and glucagon, as also appears to be the case with hepatic carbohydrate metabolism.

A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis.

Studied on the oxidation of oleic and octanoic acids to ketone bodies were carried out in homogenates and in mitochondrial fractions of livers taken from fed and fasted rats. Malonyl-CoA inhibited ketogenesis from the former but not from the latter substrate. The site of inhibition appeared to be the carnitine acyltransferase I reaction. The effect was specific and easily reversible. Inhibitory concentrations were in the range of values obtained in livers from fed rats by others. It is proposed that malonyl-CoA functions as both precursor for fatty acid synthesis and suppressor of fatty acid oxidation. As such, it might be an important element in the carbohydrate-induced sparing of fatty acid oxidation.

In vitro reversal of the fasting state of liver metabolism in the rat. Reevaluation of the roles of insulin and glucose.

Studies were conducted to determine whether the direction of hepatic carbohydrate and lipid metabolism in the rat could be switched simultaneously from a "fasted" to a "fed" profile in vitro. When incubated for 2 h under appropriate conditions hepatocytes from fasted animals could be induced to synthesize glycogen at in vivo rates. There was concomitant marked elevation of the tissue malonyl-coenzyme A level, acceleration of fatty acid synthesis, and suppression of fatty acid oxidation and ketogenesis. In agreement with reports from some laboratories, but contrary to popular belief, glucose was not taken up efficiently by the cells and was thus a poor substrate for eigher glycogen synthesis or lipogenesis. The best precursor for glycogen formation was fructose, whereas lactate (pyruvate) was most efficient in lipogenesis. In both case the addition of glucose to the gluconeogenic substrates was stimulatory, the highest rates being obtained with the further inclusion of glutamine. Insulin was neither necessary for, nor did it stimulate, glycogen deposition or fatty acid synthesis under favorable substrate conditions. Glucagon at physiological concentrations inhibited both glycogen formation and fatty acid synthesis. Insulin readily reversed the effects of glucagon in the submaximal range of its concentration curve. The following conclusions were drawn. First, the fasted-to-fed transition of hepatic carbohydrate and lipid metabolism can be accomplished in vitro over a time frame similar to that operative in vivo. Second, reversal appears to be a substrate-driven phenomenon, in that insulin is not required. Third, unless an unidentified factor (present in protal blood during feeding) facilitates the uptake of glucose by liver it seems unlikely that glucose is the immediate precursor for liver glycogen or fat synthesis in vivo. A likely candidate for the primary substrate in both processes is lactate, which is rapidly formed from glucose by the small intestine and peripheral tissues. Fructose and amino acids may also contribute. Fourth, the requirement for insulin in the reversal of the fasting state of liver metabolism in vivo can best be explained by its ability to offset the catabolic actions of glucagon.

Time course and significance of changes in hepatic fructose-2,6-bisphosphate levels during refeeding of fasted rats.

The time course of changes in hepatic fructose-2,6-bisphosphate (F-2,6-P2) and glycogen content was examined in fasted rats infused with glucose intragastrically or allowed to eat a chow diet ad lib. Initial values for the two parameters were approximately 0.4 nmol/g and 2 mg/g of tissue, respectively. Contrary to what might have been expected on the basis of reported studies with hepatocytes exposed to glucose (i.e., a rapid elevation of F-2,6-P2), the rise in F-2,6-P2 levels in vivo was a late event. It began only 4-5 h after glucose administration or refeeding, at which time glycogen content had reached approximately 35 mg/g of tissue. Thereafter, [F-2,6-P2] climbed rapidly, attaining fed values in the region of 10 nmol/g as glycogen stores became maximal (approximately 60 mg/g of tissue). Although the biochemical basis for these changes is still unclear, the delayed increase in [F-2,6-P2] is entirely consistent with the fact that much of the glycogen deposited in liver in the early postprandial phase is gluconeogenic in origin. The later rise in [F-2,6-P2] likely represents a key signal for the attenuation of gluconeogenic carbon flow into glycogen as the latter approaches repletion levels.

Exercise intolerance, lactic acidosis, and abnormal cardiopulmonary regulation in exercise associated with adult skeletal muscle cytochrome c oxidase deficiency.

A 27-yr-old woman with lifelong severe exercise intolerance manifested by muscle fatigue, lactic acidosis, and prominent symptoms of dyspnea and tachycardia induced by trivial exercise was found to have a skeletal muscle respiratory chain defect characterized by low levels of reducible cytochromes a + a3 and b in muscle mitochondria and marked deficiency of cytochrome c oxidase (complex IV) as assessed biochemically and immunologically. Investigation of the pathophysiology of the exercise response in the patient revealed low maximal oxygen uptake (1/3 that of normal sedentary women) in cycle exercise and impaired muscle oxygen extraction as indicated by profoundly low maximal systemic arteriovenous oxygen difference (5.8 ml/dl; controls = 15.4 +/- 1.4, mean +/- SD). The increases in cardiac output and ventilation during exercise, normally closely coupled to muscle metabolic rate, were markedly exaggerated (more than two- to threefold normal) relative to oxygen uptake and carbon dioxide production accounting for prominent tachycardia and dyspnea at low workloads. Symptoms in our patient are similar to those reported in other human skeletal muscle respiratory chain defects involving complexes I and III, and the exaggerated circulatory response resembles that seen during experimental inhibition of the mitochondrial respiratory chain. These results suggest that impaired oxidative phosphorylation in working muscle disrupts the normal regulation of cardiac output and ventilation relative to muscle metabolic rate in exercise.

Characteristics of fatty acid oxidation in rat liver homogenates and the inhibitory effect of malonyl-CoA.

Experiments were carried out to study the control of fatty acid oxidation and ketogenesis in rat liver homogenates. In contrast to findings with the perfused liver, rates of fatty acid oxidation were high and equal in liver homogenates from fed and fasted animals. No difference in apparent Km values for oleate, ATP, coenzyme A or carnitine could be detected in the two types of homogenate. Over the concentration range 20--40 micron, malonyl-CoA inhibited oleate oxidation by 50--75%. The fact that the inhibitory effect could be removed by pre-treatment with alkali or fatty acid synthetase indicated that the inhibitory molecule was malonyl-CoA rather than a contaminant. The effect was readily reversible and appeared to be competitive with oleyl-CoA. Malonyl-CoA also inhibited oleate oxidation in homogenates of heart and kidney cortex but this is unlikely to have physiological relevance since, in contrast to liver, neither tissue contains an active cytosolic pathway for the generation of malonyl-CoA and the synthesis of fatty acids.

Hepatic fatty acid oxidation and ketogenesis after clofibrate treatment.

The effect of clofibrate treatment on hepatic ketogenic capacity was studied in rats. Ketogenesis from octanoate and oleate was increased 2- and 4,5-fold, respectively, in hepatocytes from fed, treated rats. In contrast to controls ketogenic rates did not increase upon starvation. While ketogenesis from oleate was higher in fed, treated animals than in fasted controls, endogenous ketogenesis was lower and increased upon starvation. Ketogenesis from octanoate and oleate was stimulated approx. 2-fold in homogenates from treated animals. Labeled pyruvate and succinate oxidation was unaltered. [1-14C]Oleate oxidation was severely inhibited by cyanide, both in homogenates from controls and treated animals. Clofibrate caused a 3-fold increase in hepatic carnitine levels. Catalase and glutamate dehydrogenase activities were also increased by the drug. Cytochrome c oxidase did not change. Despite their increased ketogenic capacity hepatocytes from treated rats esterified as much oleate as controls. The increased oxidation was matched by an increased oleate uptake. Plasma ketones were increased 2-fold in fasted, treated animals. Plasma free fatty acids were unaffected. It is concluded that the enhanced ketogenic capacity induced by clofibrate is the result of an increase in mitochondrial beta-oxidation, an increase in the activity of carnitine palmitoyltransferase and possibly of the observed increases in hepatic carnitine content and fatty acid uptake.

Effects of exogenous fatty acid concentration on glucagon-induced changes in hepatic fatty acid metabolism.

Studies were conducted to clarify the relationship between the external fatty acid concentration and glucagon in the regulation of hepatic fatty acid metabolism. Hepatocytes from fed rats were incubated with increasing concentrations of oleate (up to 1 mM) in the presence and absence of glucagon and the time sequence of changes in cellular malonyl-CoA levels, fatty acid synthesis, fatty acid oxidation, and ketogenesis were measured. At low concentrations of fatty acid the effect of glucagon was to abolish malonyl-CoA synthesis and lipogenesis and to produce a marked stimulation of fatty acid oxidation and ketogenesis. Similar effects were obtained with high concentrations of fatty acid in the absence of glucagon and, under these conditions, the additional presence of the hormone produced little further response. The results are consistent with the concept that the rate of fatty acid oxidation in liver is dictated largely by the relative concentrations of long-chain acyl-CoA (substrate for carnitine acyltransferase I) and malonyl-CoA (inhibitor of the transferase). They also indicate that the preemptive effect of fatty acids on glucagon-induced changes in fatty acid metabolism stems from their ability to reduce the tissue malonyl-CoA content, probably through long-chain acyl-CoA suppression of acetyl-CoA carboxylase.

Evidence for suppression of hepatic glucose-6-phosphatase with carbohydrate feeding.

The mechanism by which exogenous glucose stimulates the incorporation of hepatic glucose-6-phosphate into glycogen in fasted rats has not been clearly delineated. We gave glucose intragastrically over a 3.5-h period during which liver glycogen was deposited at linear rates. Simultaneous primed continuous infusion of [2-3H] or [3-3H]glucose established that under these conditions absolute carbon flow through hepatic glucose-6-phosphatase was greatly suppressed. After 1 h, hepatic [UDP-glucose] and [glucose-6-phosphate] had fallen by 50-60% and the former remained low throughout the experiment. By contrast, [glucose-6-phosphate] rebounded to its initial value by 2 h and remained at this level during the subsequent hour. We interpret the data as follows. Exogenous glucose, in addition to acting as a precursor of liver glucose-6-phosphate, causes diversion of the latter away from free glucose formation and into glycogen synthesis. The fall in [UDP-glucose] is in accord with a glucose-induced activation of glycogen synthase, as proposed by Hers (Annu. Rev. Biochem. 1976; 45:167-89.). However, the fall-rise sequence of glucose-6-phosphate concentration constitutes the first direct evidence in vivo for simultaneous inhibition at the level of glucose-6-phosphatase.

More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling.

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic beta-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802-5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose-->malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal control of ketogenesis. Biochemical considerations.

A two-site, bihormonal concept for the control of ketone body production is proposed. Thus, ketosis is viewed as the result of increased mobilization of free fatty acids from adipose tissue (site 1) to the liver (site 2), coupled with simultaneous enhancement of the liver's capacity to convert these substrates into acetoacetic and beta-hydroxybutyric acids. The former event is believed to be triggered by a fall in plasma insulin levels while the latter is considered to be effected primarily by the concomitant glucagon excess characteristic of the ketotic state. Although the precise mechanism whereby elevation of the circulating [glucagon]:[insulin] ratio stimulates hepatic ketogenic potential is not known, activation of the carnitine acyltransferase reaction, the first step in the oxidation of fatty acids, is an essential feature. Two prerequisites for this metabolic adaptation in liver appear to be an elevation in its carnitine content and depletion of its glycogen stores. Despite present limitations the model (evolved mainly from rat studies) provides a framework for the description of various types of clinical ketosis in biochemical terms and may be useful for future studies.

Kinetics of carnitine-dependent fatty acid oxidation: implications for human carnitine deficiency.

The relationship between the concentration of carnitine and the oxidation of oleate was examined in homogenates prepared from skeletal muscle, liver, kidney, and heart of the rat, and from canine and human skeletal muscle. the carnitine content of these tissues in situ spanned a wide range, from about 0.1 mumol per gram in rat liver to about 3.0 mumol per gram in human muscle. The concentration of carnitine required for half-maximal rates of fatty acid oxidation in vitro also varied greatly (10 to 15 microM for rat liver to 200 to 400 microM for human muscle), and in rough proportion to the normal carnitine content of the tissues. For any given tissue, the carnitine content seems to be set at a level necessary for optimal rates of fatty acid oxidation. The data provide a plausible explanation for the fact that muscle fatty acid metabolism is severely impaired in the syndrome of human carnitine deficiency, since measured carnitine levels are in the range expected to limit substantially the capacity for fatty acid oxidation.

New insights into the mitochondrial carnitine palmitoyltransferase enzyme system.

Dissection of the mitochondrial carnitine palmitoyltransferase (CPT) enzyme system in terms of its structure/function relationships has proved to be a formidable task. Although no one formulation has gained universal agreement we believe that the weight of evidence supports a model with the following features: a) in any given tissue CPT I and CPT II are distinct proteins; b) CPT I, unlike CPT II, is detergent labile; c) within a species CPT II is expressed body wide, whereas CPT I exists as tissue specific isoforms; d) malonyl-CoA and other CPT I inhibitors probably interact at the catalytic center of the enzyme, not with a regulatory subunit. The amino acid sequences of rat and human CPT II (deduced from cDNA clones) show them to be similar proteins (greater than 80% identity) but encoded by mRNAs of significantly different sizes. Efforts to clone and sequence the cDNA for rat liver CPT I are presently underway.

Regulation of lipoprotein lipase in different rat tissues.

In rats maintained on a fat-free diet, hepatic production of very low density lipoprotein (VLDL) fell by only 18% after 20 hours of fasting (as judged by rates of triglyceride accumulation in plasma following administration of Triton WR1339). Infusion of heparin into fed rats caused a rapid accumulation in plasma of two distinct lipase activities in approximately equal amounts: heparin releasable lipoprotein lipase (HR-LPL) and heparin releasable triglyceride lipase (HR-TGL). Fasting reduced the former by 67%, but the latter by only 20 to 25%. To gain insight into the effects of nutritional status on the interorgan distribution of these enzymes, experiments were designed for the perfusion of liver and heart by standard procedures, and of epididymis, gastrocnemius muscle (red fiber), and psoas muscle (white fiber) by micro techniques. After addition of heparin, perfusates were analyzed for each species of lipase. The objective was to selectively monitor the capillary-bound, physiologically functional lipase without significant contamination by intracellular enzyme. HR-TGL was found only in liver, where it represented the major lipase activity; it was only minimally affected by fasting. In epididymis HR-LPL fell by 85% in fasted animals and, remarkably, rebounded almost to ad libitum fed values after only two hours of refeeding. In both heart and gastrocnemius muscle, directional changes in HR-LPL were exactly reciprocal to those seen in epididymis. By contrast, HR-LPL of psoas muscle was unresponsive to fasting. Qualitatively, the data were in keeping with documented nutritionally-induced shifts in tissue disposal of VLDL-triglycerides.(ABSTRACT TRUNCATED AT 250 WORDS)

[To visualize the posterolateral segment of the external meniscus. Value of the position of internal flexion rotation]. / Visualiser le segment postéro-latéral du ménisque externe. Intérêt de la position en flexion rotation interne.

Interpretation of images of the posterolateral segment of external meniscus, during arthrography with contrasts of knee, is known to be difficult and this explains the possibility of false positives and the frequency of false negatives observed during exploration on this meniscus. A flexion-internal rotation projection of knee was used to improve the image of the posterior horn of the external meniscus, and analysis of results of about 100 examinations showed significant benefits. Use of the method is illustrated in a normal knee, and one with a rupture of posterior horn of external meniscus masked in extension and detected only in the flexion-internal rotation projection.

[A new technique of pulmonary arterial angioscopy: capnoangioscopy. An experimental study]. / Nouvelle technique d'angioscopie artérielle pulmonaire: la capnoangioscopie. Etude expérimentale.

From animal experiments (n = 10), the authors describe a new technique of pulmonary arterial angioscopy. A balloon-fitted catheter is introduced into the trunk of one of the pulmonary arteries. A small caliber angioscope with an end capable of being propped up is then pushed within the lumen of the catheter. Intraluminal visualization of the pulmonary artery and its branches cannot begin until after the balloon is inflated to arrest blood flow and the visualization chamber is cleared by sterile carbon dioxide. The quality of the image obtained is similar to that of bronchial endoscopy, making is possible to examine at length the walls of the proximal and distal pulmonary arteries up to a caliber of 2.5 mm, and to detect blood clots. Pulmonary angioscopy is tolerated to the same extent as right heart catheterization. Potential applications of this technique are diagnosis of chronic cor pulmonale and treatment of pulmonary embolism.