Inhibition of muscular ketone extraction by lipid infusion in man

Using the forearm technique, muscular ketone body metabolism was investigated in 12 healthy volunteers during an i.v. infusion of lipid emulsions containing long chain triglycerides (LCT) or a mixture of medium- and long chain triglycerides (MCT/LCT). During the basal period, arterial concentrations and muscular extraction of beta-hydroxybutyrate and acetoacetate were linearly correlated as expected. This relationship was abolished during the infusion of both lipid emulsions. In addition, fractional extraction rates of ketone bodies were reduced. These changes were most probably mediated by elevated levels of free fatty acids and triglycerides as well.     

Evidence for a substrate regulation of triglyceride lipolysis in human skeletal muscle

Glycerol release from the human forearm which is generally used as a semiquantitative index of intramuscular lipolysis was studied under different hormonal influence and substrate supply in healthy volunteers and juvenile diabetics using the forearm technique. Acute insulin deficiency in juvenile diabetics failed stimulating the rate of muscular lipolysis since the rates of glycerol release in normals and diabetics were the same. In addition, in normal volunteers high physiological levels of insulin caused by an intraarterial infusion of the hormone exhibited no effect on the glycerol release from deep forearm tissue. Similarly, an intraarterial infusion of metaproterenol did not accelerate muscular glycerol release in normal man. However, in juvenile diabetics in acute insulin deficiency the same dose of the catecholamine increased the rate of muscular glycerol production. Elevated substrate supply during intravenous infusion of glucose or fructose yielded increased uptake of glucose and fructose into the deep forearm tissue and thereby promptly blocked muscular glycerol release in normal volunteers and in juvenile diabetics. These findings suggest that the rate of lipolysis in muscle tissue is not primarily under the control of hormones but rather by substrate supply.     

Inhibition of muscular triglyceride lipolysis by ketone bodies: a mechanism for energy-preservation in starvation

Using the forearm technique, muscle substrate balances were measured in healthy volunteers under the influence of a low dose intraarterial infusion of beta-hydroxybutyrate. The ketone body led to a prompt cessation of muscular release of glycerol and free fatty acids, indicating inhibited muscular triglyceride lipolysis. This finding confirms the concept of substrate regulation of muscle triglyceride lipolysis in vivo.     

Metabolic studies during normoglycaemic clamping of insulin-dependent diabetics using a glucose-controlled insulin infusion system.

Metabolic rhythms have been studied in six insulin-dependent diabetics during subcutaneous insulin therapy, and during control of blood glucose concentration by a glucose-controlled insulin infusion system (GCIIS). In none of the subjects was blood glucose concentration consistently within the normal range during subcutaneous insulin therapy. In contrast, blood glucose concentration was within the normal range after 3.5 h of insulin delivery by the glucose-controlled insulin infusion system and remained in the normal range for the following 8 h through lunch and dinner. Mean blood glucose concentration during this time ranged from 5.31 to 7.90 mM. Following normalisation of blood glucose concentration, blood lactate and pyruvate were similar with both the GCIIS and subcutaneous insulin therapy. Post-prandial lactate peaks were delayed with the GCIIS. Alanine levels were consistently higher during control with the GCIIS compared with subcutaneous therapy, while blood ketone body and plasma NEFA levels were lower, and the premeal peaks in the lipid metabolites were delayed. It is not possible to conclude that attainment of normoglycaemia with the present generation of glucose-controlled insulin infusion systems in insulin-dependent diabetics is accompanied by total normalisation of intermediary metabolism.     

Ketone body kinetics in humans: the effects of insulin-dependent diabetes, obesity, and starvation

The kinetics of acetoacetate (A) and beta-hydroxybutyrate (B) have been studied following the injection as a pulse or continued infusion of [3-14C]acetoacetate (A*) or [14C]beta-hydroxybutyrate (B*) into six newly diagnosed, untreated, ketotic diabetic patients, ten obese subjects in the postabsorptive state, and the ten obese subjects after 1-2 weeks starvation (50 cal per day). Employing a compartmental model of acetoacetate and beta-hydroxybutyrate kinetics developed using CONSAM for normal subjects, the rate coefficients (Lij), rates of release of newly synthesized acetoacetate and beta-hydroxybutyrate into the blood (UA, UB), and fractional removal of each compound (FCRA and FCRB) were calculated. Ketone body release into blood (UA + UB) in diabetic subjects was threefold higher than normal (mean +/- SD, 208 +/- 118 versus 81 +/- 66 mumol min-1 m-2) and in obese subjects the rate increased on starvation from 171 +/- 70 to 569 +/- 286 mumol min-1 m-2. In each case most of the increase was in beta-hydroxybutyrate. The major change in diabetes and on starvation of the obese subjects was in the rate coefficient for removal of ketone bodies. Normally 0.168 +/- 0.109 min-1, it was 0.055 +/- 0.040 min-1 in the diabetic patients and fell from 0.066 +/- 0.040 to 0.027 +/- 0.019 min-1 in the obese subjects on starvation. In normal subjects, FCRA was similar to FCRB (0.226 +/- 0.142 versus 0.188 +/- 0.124 min-1). However, in diabetics, FCRA was 0.074 +/- 0.044 and FCRB was 0.050 +/- 0.034 min-1 and both were lower than normal. On starvation of obese subjects, FCRA fell from 0.199 +/- 0.047 to 0.089 +/- 0.035 min-1, whereas FCRB fell from 0.141 +/- 0.040 to 0.033 +/- 0.012 min-1. Therefore, the removal of beta-hydroxybutyrate was impaired more than that of acetoacetate in all patients. Our results confirm previous observations that ketosis is associated with high rates of ketogenesis and a decrease in fractional clearance. In addition, we found that in diabetes, obesity, and in obese subjects following starvation, most of the increased synthesis was in beta-hydroxybutyrate and that the clearance of beta-hydroxybutyrate decreased more than that of acetoacetate.     

Ketogenic effects of carnitine in patients with muscular dystrophy and cytochrome oxidase deficiency

The effects of a single oral dose of carnitine on fasting-induced ketosis was investigated in four normal individuals, five patients with muscular dystrophy, and one patient with a generalized cytochrome c oxidase deficiency. Plasma carnitine, free fatty acids, glucose, insulin, and glucagon were also measured. Normal individuals showed an average 0.09 mM increase in blood beta-hydroxybutyrate concentration during a 12- to 18-hr period of fasting and carnitine administration did not affect this response (average: 0.12 mM). Muscular dystrophy patients showed a greater fasting-induced elevation in beta-hydroxybutyrate (average 0.29 mM) and carnitine administration greatly enhanced this ketogenic response (average 0.84 mM). The cytochrome c oxidase deficient patient showed an even larger increase in beta-hydroxybutyrate with fasting (1.67 mM) and carnitine further augmented this ketotic effect (3.78 mM). Plasma free fatty acids were also elevated in patients that showed enhanced ketosis. Plasma glucagon concentration did not change, but insulin levels decreased during the 12- to 18-hr period of fasting; no major differences were found between controls and patients. These results indicate that some patients with muscular dystrophy and cytochrome c oxidase deficiency are more prone to develop ketosis than normal individuals and that carnitine administration enhances this response. Since both muscular dystrophy patients and the patient with cytochrome c oxidase deficiency had similar ketogenic responses, the data suggest that ketone body utilization may be impaired in these patients. The ability of L-carnitine to be ketogenic should be considered in the treatment of these patients.     

Muscle metabolism during exercise in diabetics and in obese during starvation

The influence of exercise on forearm muscle metabolism was examined in 9 healthy subjects, in 16 diabetics and in 4 obese subjects during complete starvation. During exercise glucose uptake rose 7-8 fold in the controls. However, no increase of glucose uptake was observed in the other groups studied. Moreover, a glucose production from the working muscle took place in about 40 percent of both the diabetic patients and the starved obese subjects. The nonutilization of glucose during physical work in the diabetic like states was accompanied by a significantly diminished lactate output. The arterial concentration of FFA, glycerol beta-HOB and Acac was markedly elevated in the starved obese patients. The FFA-uptake at rest and during exercise, however, was not different from results of controls. Whereas an effux of beta-HOB has been observed during exercise, Acac uptake was increased in these patients. It is suggested that in maturity onset and starvation diabetes glycolysis is inhibited.     

Formation of free acetate by isolated perfused livers from normal, starved and diabetic rats

Isolated rat livers perfused in an open system exhibited a continous net release of free acetate. Upon intraportal infusion of hexanoate the net release of total ketone bodies and of free acetate increased significantly in livers from fed and 48 hours starved rats. The ratio ketone body production/acetate production during infusion of hexanoate was similar with livers from fed and starved rats. Livers from diabetic rats, however, did not only exhibit a higher rate of ketone body and acetate production, but also a significant decrease of the ratio ketone body production/acetate production. Intraportal infusion of oleate led also to an enhanced release of free acetate. An examination of the activities of 5 enzymes involved in ketone body and acetate metabolism showed no correlation with the higher rate of acetate production by diabetic livers.     

Novel aspects of the activities and subcellular distribution of enzymes of ketone body metabolism in the liver and kidney of the goldfish, Carassius auratus

The metabolic organization of ketone body metabolism of liver and kidney of the goldfish Carassius auratus was assessed by measuring maximal activities, subcellular distribution, and stereoisomer preference of ketone body enzymes. These determinations indicate that the organization of ketone body metabolism in liver and kidney of goldfish differs from that of mammals in some respects. All the enzymes of ketone body metabolism were present in liver and kidney of goldfish, with the exception of hydroxymethylglutaryl-CoA (HMG-CoA) synthetase, which was not detected in liver. Two forms of beta-hydroxybutyrate dehydrogenase (betaHBDH) with different stereospecificity for beta-hydroxybutyrate (D- and L-beta-hydroxybutyrate) were detectable in liver and kidney. All of the ketone body enzymes measured in liver were mainly in the mitochondrial fraction, with the exception of D- and L-betaHBDH, which were cytosolic. In kidney, HMG-CoA synthase, together with HMG-CoA lyase and acetoacetyl CoA thiolase (AcoAT), were found mainly in the mitochondrial fraction. L-betaHBDH was mainly cytosolic in kidney, but by contrast with liver, D-betaHBDH was mainly found in the mitochondria, and SKT was distributed in both the mitochondrial and cytosolic compartments. J. Exp. Zool. 286:434-439, 2000.     

Metabolic characteristics of human subcutaneous abdominal adipose tissue after overnight fast

Subcutaneous abdominal adipose tissue is one of the largest fat depots and contributes the major proportion of circulating nonesterified fatty acids (NEFA). Little is known about aspects of human adipose tissue metabolism in vivo other than lipolysis. Here we collated data from 331 experiments in 255 healthy volunteers over a 23-year period, in which subcutaneous abdominal adipose tissue metabolism was studied by measurements of arterio-venous differences after an overnight fast. NEFA and glycerol were released in a ratio of 2.7:1, different (P < 0.001) from the value of 3.0 that would indicate no fatty acid re-esterification. Fatty acid re-esterification was 10.2 ± 1.4%. Extraction of triacylglycerol (TG) (fractional extraction 5.7 ± 0.4%) indicated intravascular lipolysis by lipoprotein lipase, and this contributed 21 ± 3% of the glycerol released. Glucose uptake (fractional extraction 2.6 ± 0.3%) was partitioned around 20-25% for provision of glycerol 3-phosphate and 30% into lactate production. There was release of lactate and pyruvate, with extraction of the ketone bodies 3-hydroxybutyrate and acetoacetate, although these were small numerically compared with TG and glucose uptake. NEFA release (expressed per 100 g tissue) correlated inversely with measures of fat mass (e.g., with BMI, r(s) = -0.24, P < 0.001). We examined within-person variability. Systemic NEFA concentrations, NEFA release, fatty acid re-esterification, and adipose tissue blood flow were all more consistent within than between individuals. This picture of human adipose tissue metabolism in the fasted state should contribute to a greater understanding of adipose tissue physiology and pathophysiology.     

Changes in acetoacetate/beta-hydroxybutyrate ratio in arterial blood following hepatic artery embolization in man

Acetoacetate/beta-hydroxybutyrate ratio in the hepatic venous blood was compared to the ratios in arterial blood and peripheral venous blood in hypoxic state following right hepatic artery embolization in 5 patients with liver cancer. Ketone body ratios in right hepatic venous blood were positively correlated with those in arterial blood (r = 0.960, p less than 0.001), but not with those in peripheral venous blood. The free NAD+/NADH ratio of the liver mitochondria, which is reflected by the ketone body ration in hepatic venous blood, can be evaluated by the ketone body ratio in the arterial blood.     

High beta-hydroxybutyrate concentration in liver and skeletal muscle of newly hatched chicks

Characteristic changes in ketone body concentrations in blood, liver, and skeletal muscle were investigated in detail in newly hatched chicks. The concentration of beta-hydroxybutyrate in the blood was maximal at hatch (0 day), markedly decreased to 3 days, then maintained at low levels, up to 14 days of age. The concentration of acetoacetate in blood, on the other hand, did not change after hatching but remained lower than that of beta-hydroxybutyrate at all ages. In liver and muscles, the concentration of beta-hydroxybutyrate changed in a manner similar to that in the blood. The muscle to blood ratio of the beta-hydroxybutyrate concentration on days -1 and 0 was significantly higher than those at 1 through 14 days post-hatch. These results show that newly hatched chicks have the same high ketone body concentrations in the skeletal muscle, blood and liver. It is, hence, suggested that uptake of beta-hydroxybutyrate by muscles is substantial or that ketogenesis, if any, occurs in muscles immediately before and after hatching of chicks.     

Evidence for a participation of the kallikrein-kinin system in the regulation of muscle metabolism during hypoxia.

The effect of hypoxia on muscle metabolism was studied in the human forearm by the registration of arterial-deep venous concentration differences of oxygen, glucose, lactate, pyruvate, acetoacetate, and muscular blood flow after short, transient arrest of the forearm circulation. These studies were performed during the intravenous infusion of physiological saline (n=4), of a kallikrein-trypsin inhibitor (n=4), and of kallikrein-trypsin inhibitor plus the intrabrachial-arterial infusion of bradykinin (n=4). Infusion of the kallikrein-trypsin inhibitor significantly reduced the well known hypoxia-induced acceleration of nuscular glucose uptake due to a reduction of blood flow and of muscular glucose extraction. These changes of muscular glucose metabolism were accompanied by more or less striking effects on the balances of oxygen, lactate and acetoacetate. Physiological doses of bradykinin into the brachial artery during the infusion of a kallikrein-trypsin inhibitor restored almost completely the metabolic response during hypoxia. From these data there is further evidence for a participation of the kallikrein-kinin system in the physiological regulation of muscular substrate metabolism.     

Effect of lactate and beta-hydroxybutyrate infusions on brain metabolism in the fetal sheep

Brain uptake of substrates other than glucose has been demonstrated in neonatal but not fetal animals in vivo. This study was undertaken to investigate the ability of the fetal sheep brain to use potential alternative substrates when they were provided in increased amounts. Brain substrate uptake was measured in chronically catheterised fetal sheep during 2-h infusions of neutralised lactate (n = 12) or beta-hydroxybutyrate (n = 12). Despite large increases in fetal arterial lactate and beta-hydroxybutyrate during the respective infusions, no significant uptake of either substrate was demonstrated. However during both types of infusion, the brain arterio-venous difference for glucose decreased 30% (P less than 0.05). Since the brain arterio-venous difference for oxygen was unchanged, and blood flow to the cerebral hemispheres (measured in 11 studies) was also unchanged, the infusions appeared to cause a true decrease in brain glucose uptake. This decrease paralleled the rise in lactate concentration during lactate infusions, and the rise in lactate and butyrate concentrations during the butyrate infusions. Both substrates have metabolic actions that may inhibit brain glucose uptake. We speculate that the deleterious effects of high lactate and ketone states in the perinatal period may in part be due to inhibition of brain glucose uptake.     

On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans

During the fasting state, insulin reduces nonesterified fatty acid (NEFA) appearance in the systemic circulation mostly by suppressing intracellular lipolysis in the adipose tissue. In the postprandial state, insulin may also control NEFA appearance through enhanced trapping into the adipose tissue of NEFA derived from intravascular triglyceride lipolysis. To determine the contribution of suppression of intracellular lipolysis in the modulation of plasma NEFA metabolism by insulin during enhanced intravascular triglyceride lipolysis, 10 healthy nonobese subjects underwent pancreatic clamps at fasting vs. high physiological insulin level with intravenous infusion of heparin plus Intralipid. Nicotinic acid was administered orally during the last 2 h of each 4-h clamp to inhibit intracellular lipolysis and assess insulin's effect on plasma NEFA metabolism independently of its effect on intracellular lipolysis. Stable isotope tracers of palmitate, acetate, and glycerol were used to assess plasma NEFA metabolism and total triglyceride lipolysis in each participant. The glycerol appearance rate was similar during fasting vs. high insulin level, but plasma NEFA levels were significantly lowered by insulin. Nicotinic acid significantly blunted the insulin-mediated suppression of plasma palmitate appearance and oxidation rates by approximately 60 and approximately 70%, respectively. In contrast, nicotinic acid did not affect the marked stimulation of palmitate clearance by insulin. Thus most of the insulin-mediated reduction of plasma NEFA appearance and oxidation can be explained by suppression of intracellular lipolysis during enhanced intravascular triglyceride lipolysis in healthy humans. Our results also suggest that insulin may affect plasma NEFA clearance independently of the suppression of intracellular lipolysis.     

Brain uptake and metabolism of ketone bodies in animal models

As a consequence of the high fat content of maternal milk, the brain metabolism of the suckling rat represents a model of naturally occurring ketosis. During the period of lactation, the rate of uptake and metabolism of the two ketone bodies, beta-hydroxybutyrate and acetoacetate is high. The ketone bodies enter the brain via monocarboxylate transporters whose expression and activity is much higher in the brain of the suckling than the mature rat. beta-Hydroxybutyrate and acetoacetate taken up by the brain are efficiently used as substrates for energy metabolism, and for amino acid and lipid biosynthesis, two pathways that are important for this period of active brain growth. Ketone bodies can represent about 30-70% of the total energy metabolism balance of the immature rat brain. The active metabolism of ketone bodies in the immature brain is related to the high activity of the enzymes of ketone body metabolism. Thus, the use of ketone bodies by the immature rodent brain serves to spare glucose for metabolic pathways that cannot be fulfilled by ketones such as the pentose phosphate pathway mainly. The latter pathway leads to the biosynthesis of ribose mandatory for DNA synthesis and NADPH which is not formed during ketone body metabolism and is a key cofactor in lipid biosynthesis. Finally, ketone bodies by serving mainly biosynthetic purposes spare glucose for the emergence of various functions such as audition, vision as well as more integrated and adapted behaviors whose appearance during brain maturation seems to critically relate upon active glucose supply and specific regional increased use.     

Ketone body kinetics in normal,diabetic, and obese humans

We have developed a model for the kinetics of acetoacetate (A) and β-OH-butyrate (B) in normal subjects. The model contains separate compartments for blood A, B, and acetone, as well as three exchange compartments. By using the model, the synthesis, utilization, and clearance rates of A and B were determined separately. We have compared the model with others that have been proposed for ketone body metabolism and have used the model to analyse studies undertaken in newly diagnosed diabetic patients and obese subjects (before and after a 2 week period of starvation). We found that in diabetic and obese individuals the synthesis of ketone bodies was higher than normal and that the fractional losses of A and B were reduced. The results suggest that ketosis develops as a result of high synthesis rates coupled with decreased fractional loss of ketone bodies. In each group the metabolism of B was altered more than A.     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

Ketone bodies alter dinitrophenol-induced glucose uptake through AMPK inhibition and oxidative stress generation in adult cardiomyocytes

In aerobic conditions, the heart preferentially oxidizes fatty acids. However, during metabolic stress, glucose becomes the major energy source, and enhanced glucose uptake has a protective effect on heart function and cardiomyocyte survival. Thus abnormal regulation of glucose uptake may contribute to the development of cardiac disease in diabetics. Ketone bodies are often elevated in poorly controlled diabetics and are associated with increased cellular oxidative stress. Thus we sought to determine the effect of the ketone body beta-hydroxybutyrate (OHB) on cardiac glucose uptake during metabolic stress. We used 2,4-dinitrophenol (DNP), an uncoupler of the mitochondrial oxidative chain, to mimic hypoxia in cardiomyocytes. Our data demonstrated that chronic exposure to OHB provoked a concentration-dependent decrease of DNP action, resulting in 56% inhibition of DNP-mediated glucose uptake at 5 mM OHB. This was paralleled by a diminution of DNP-mediated AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation. Chronic exposure to OHB also increased reactive oxygen species (ROS) production by 1.9-fold compared with control cells. To further understand the role of ROS in OHB action, cardiomyocytes were incubated with H(2)O(2). Our results demonstrated that this treatment diminished DNP-induced glucose uptake without altering activation of the AMPK/p38 MAPK signaling pathway. Incubation with the antioxidant N-acetylcysteine partially restored DNP-mediated glucose but not AMPK/p38 MAPK activation. In conclusion, these results suggest that ketone bodies, through inhibition of the AMPK/p38 MAPK signaling pathway and ROS overproduction, regulate DNP action and thus cardiac glucose uptake. Altered glucose uptake in hyperketonemic states during metabolic stress may contribute to diabetic cardiomyopathy.     

The regulation of glutamine and ketone-body metabolism in the small intestine of the long-term (40-day) streptozotocin-diabetic rat.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.