Enhancement of mitochondrial carnitine and carnitine acylcarnitine translocase-mediated transport of fatty acids into liver mitochondria under ketogenic conditions.

Carnitine acylcarnitine translocase of rat liver mitochondria, assayed by following the uptake of radioactive carnitine, resembled the translocase of heart mitochondria described earlier (Pande, S. V., and Parvin, R. (1976) J. Biol. Chem. 251,6683-6691) in that it showed first order kinetics for the initial uptake and a marked sensitivity to inhibition by mersalyl, N-ethylmaleimide, and (+)-decanoylcarnitine. (-)-Carnitine and its esters were transported more actively than were the corresponding (+)-carnitines. Fasting and alloxan diabetes enhanced the rates of carnitine acylcarnitine translocase-catalyzed transport of carnitine in liver mitochondria. Measurements of K, for carnitine transport and intramitochondrial carnitine concentrations showed that carnitine acylcarnitine translocase normally remains subsaturated with respect to the level of matrix carnitine and that the enhancement of transport on fasting and alloxan diabetes results from an elevation of the intramitochondrial carnitine. These results indicate that the ability of liver to transport fatty acids into mitochondria is increased under ketogenic conditions. The intramitochondrial carnitine content was found positively related to liver carnitine in a variety of conditions. Fasting for upto 48 h had little effect on serum total carnitine but decreased the urinary excretion of carnitine and deoxycarnitine and appeared to enhance carnitine retention in body. The ratio of esterified to free carnitine, in serum as well as urine, rose on fasting. It seems that under conditions of active hepatic fatty acid oxidation, like acetoacetate and /3-hydroxybutyrate, short chain acylcarnitines are produced in and exported out of liver to serve as fuel for extrahepatic tissues.

Carnitine acylcarnitine translocase of rat liver mitochondria, assayed by following the uptake of radioactive carnitine, resembled the translocase of heart mitochondria described earlier (Pande, S. V., and Parvin, R. (1976) J. Biol. Chem. 251,6683-6691) in that it showed first order kinetics for the initial uptake and a marked sensitivity to inhibition by mersalyl, N-ethylmaleimide, and (+)-decanoylcarnitine.
(-)-Carnitine and its esters were transported more actively than were the corresponding (+)-carnitines. Fasting and alloxan diabetes enhanced the rates of carnitine acylcarnitine translocase-catalyzed transport of carnitine in liver mitochondria. Measurements of K, for carnitine transport and intramitochondrial carnitine concentrations showed that carnitine acylcarnitine translocase normally remains subsaturated with respect to the level of matrix carnitine and that the enhancement of transport on fasting and alloxan diabetes results from an elevation of the intramitochondrial carnitine.
These results indicate that the ability of liver to transport fatty acids into mitochondria is increased under ketogenic conditions.
The intramitochondrial carnitine content was found positively related to liver carnitine in a variety of conditions. Fasting for upto 48 h had little effect on serum total carnitine but decreased the urinary excretion of carnitine and deoxycarnitine and appeared to enhance carnitine retention in body. The ratio of esterified to free carnitine, in serum as well as urine, rose on fasting. It seems that under conditions of active hepatic fatty acid oxidation, like acetoacetate and /3-hydroxybutyrate, short chain acylcarnitines are produced in and exported out of liver to serve as fuel for extrahepatic tissues.
The rates of hepatic fatty acid utilization are markedly influenced by the delivery rate of free fatty acids to liver of normal and ketotic rats alike. Livers from such rats take up similar amounts of fatty acids from perfusion medium but the subsequent pattern of intracellular fatty acid utilization differs between the two. In livers from normal fed rats most of the fatty acid taken up is directed toward triglyceride formation and only smaller amounts are oxidized. In ketotic rats the proportion of fatty acids being used for t,riglyceride synthesis * This work was supported by grants from the Medical Research Council of Canada (MT-4264) and the Quebec Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Affiliated to the University of Montreal.
is decreased while that being oxidized is increased (l-5). These observations have shown that the greater availability of free fatty acids is not the only factor important in determining the rates of ketogenesis since, in ketotic states, the inherent ability of liver to direct fatty acids to ketone bodies is increased. Recent evidences indicate that this ketogenic adaptation occurs at some carnitinel-dependent transport step of fatty acids in mitochondria (5-9) and that glucagon plays some role in this process (10-12).
Evidence that a carnitine-dependent step is a likely site of metabolic adaptation (5-9, 11, 12) rests on the following observations: (a) that such an adaptation, seen with long chain fat,ty acids, oxidation of which is carnitine-dependent, has no effect on ketogenesis from octanoic acid, oxidation of which is not obligatorily dependent on carnitine, (6) that with octanoic acid livers from fed rats produce as much ketone bodies as do livers from fasted rats with either long chain fatty acids or octanoic acid, and (c) that unlike octanoic acid when (-)-octanoylcarnitine is perfused, high rates of ketogenesis are observed only with livers of fasted and not with those of fed rats. These observations suggest that the ketogenic adaptation causes activation of some carnitine-dependent step(s) related to fatty acid transport but involved subsequent to the formation of acylcarnitine.
The possibility that the ketogenic adaptation involves enhancement of carnitine palmitoyltransferase activity has been examined and it is now believed that marginal increases in the overall activity of this enzyme in ketogenic states do not correlate well with the differences in the rates of fatty acid oxidation observed between livers of normal and ketotic rats (11,13). Recently, increases in hepatic carnitine concentrations were found in fasting (14) and diabetes (15). McGarry et al. (11) have shown further that the increases in hepatic carnitine in these conditions, as well as on glucagon infusion, correlate well with the ketogenic ability of liver and have proposed that increased carnitine levels promote ketogenesis by increasing the flux of fatty acids through the carnitine acyltransferase reaction, presumably by activating that carnitine acyltransferase which is situated on the inner side of the inner mitochondrial membrane. Their more recent observations suggest that the flux through carnitine palmitoyltransferase may be controlled by the levels of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase (16). Our findings (17, 18) and those of Ramsay and Tubbs (19) For the preparation of mitochondria, the homogenate was centrifuged for 10 min at 400 X g and the resulting supernatant at 8,700 x g for 5 min. The supernatant thus obtained along with the lightly packed layer on the surface of the pellet was aspirated off and the pellet was gently rinsed twice with a small volume of the homogenizing medium to free it from the lightly packed material. The pellet was washed by being suspended in fresh homogenizing medium followed by 5 min of centrifugation at 8,700 x g and then rinsed as described above. The rinsed pellet was suspended in fresh homogenizing medium to obtain a preparation of mitochondria. The above operations were carried out at 0-4°C.
The yield of mitochondria varied between 7 to 11 mg of protein for each gram of liver. These mitochondria showed good respiratory control ratios (7.5 t 0.3) and ADP-stimulated respiratory rates (147 + 7 natom of O/min/mg of protein at 30°C, mean rt SE., n = 6) with glutamate and malate as substrates. Small Scale Preparation of Liver Mitochondria by a Silicone Oil Method-The amount of total carnitine associated with mitochondria, isolated as above, was found to decrease gradually on successive washing of mitochondria. This loss was enhanced by warming and mechanical agitation, e.g. blending of mitochondria on a Vortex mixer, and it thus appeared that the amount of carnitine associated with conventionally isolated and washed mitochondria was likely to give an underestimate of the amount of endogenous carnitine initially present in mitochondria. To minimize such losses a more rapid method for the isolation of mitochondria was worked out. Soon after preparation of homogenate as described above, 3 ml was centrifuged at 500 x g for 60 s using a Fisher microfuge.
One and a half milliliters of the resulting supernatant was transferred to Eppendorf tubes containing 100 ~1 of a silicone oil of specific gravity 1.05 and the tubes were centrifuged for 90 s at 7,000 x g. The supernatant above the oil was aspirated off. The walls of the tube and the oil surface was washed with fresh medium. All liquid above oil and the material hanging in the oil layer was aspirated away along with part of the silicone oil. The pellet below the silicone oil was brought into suspension with 106 ~1 of 210 mru mannitol, 70 mM sucrose, CO mM Tris/HCl (pH 7.4). 0.1 mM EDTA. and gentle stirring with a glass rod. The tubes were then centrifuged at500 X g for 30 s. Any contaminating red blood cells, unbroken cells, etc., appeared below the surface of the silicone oil. The suspension above the silicone oil was separated and used as a mitochondrial preparation. Previously one of us mentioned, without presenting data, that carnitine transport in liver mitochondria seemed to proceed in a manner analogous to that in heart mitochondria (17). Suggestions have since been made that, unlike in heart, carnitine acylcarnitine translocase may not be present in liver mitochondria (20,21), presumably because liver mitochondria had once been reported (27) to have little carnitine and mitochondria of liver were used in those experiments which had earlier led to the consensus that carnitine was unable to cross the mitochondrial inner membrane (27-30). When experiments are set up to detect an exchange diffusion either by following an efflux of carnitine from mitochondria previously loaded with radioactive carnitine, under conditions previously described for heart mitochondria (17, 18, 31), or by following carnitine influx, as presently outlined, carnitine transport becomes measurable in liver as in heart mitochondria.
However, because isolated mitochondria from livers of normal rats exhibit much smaller (5 one-eighth) carnitine pool size as compared to those from heart, carnitine acylcarnitine translocase is technically less readily assayable in liver than in heart mitochondria.
Using a sensitive carnitine assay (24) we find, as described below (a) that reproducibly measurable amounts of carnitine are found associated with isolated liver mitochondria, (b) that such carnitine association appears to result largely from the presence of carnitine in mitochondrial matrix, and (c) that the exact value of mitochondrial carnitine depends on the method employed for the isolation of mitochondria as well as on the physiological state of rats. Carnitine transport in liver mitochondria, measured as carnitine influx, was inhibited by low concentrations of decanoyl(+)-carnitine, mersalyl, and N-ethylmaleimide, the same compounds previously recognized as effective inhibitors of the transport, measured as carnitine efflux, in heart mitochondria (17, 18). The concentration of these three inhibitors required for 50% inhibition (2 mg of mitochondria were incubated with the inhibitors for 60 s and then [14C]carnitine was added to initiate influx) was estimated as 1.5, 4, and 30 PM, respectively. Data of Table I with liver mitochondria show, in agreement with that described for heart earlier (17, 18) (a) that naturally occurring carnitine and its esters were more effective in transport than were the corresponding (+) isomers, (b) that low, near-physiological, concentrations of carnitine and acylcarnitines served as substrates for carnitine acylcarnitine translocase, (c) that acylation of carnitine enhanced its ability to serve as a substrate, and (d) that within the limits examined an increase in the chain length of the acyl portion of acylcarnitine further increased its effectiveness as substrate (compare data with decanoylcarnitine and acetylcarnitine at 0.05 mM). In experiments utilizing heart mitochondria and when the transport was followed as efflux of mitochondrial carnitine, carnitine acylcarnitine translocase catalyzed exchange was found to follow first order reaction kinetics (18). Fig. 1 shows that first order reaction kinetics apply for the initial influx of carnitine in liver mitochondria also. These observations together with the findings (see below, Fig. 1 Fig. 1 a marked deviation from first order reaction kinetics was observed for time points beyond 30 s (not shown). The intramitochondrial carnitine pool in freshly isolated liver mitochondria consisted of free carnitine as well as of appreciable amounts of acylated carnitine (Table II), and it is possible that different rates of influx of medium carnitine result at different time points depending upon whether external carnitine is exchanging with internal free carnitine, acylated carnitine, or a mixture of the two, composition of which is likely to change progressively with increasing influx of free carnitine. Moreover, it is likely that as with other metabolites (cfi 32) not all the intramitochon- drial carnitines equilibrate rapidly enough to constitute a homogeneous pool. On prolonging incubation for 20 min the uptake of carnitine, measured from radioactivity and taking into account the slight dilution of the specific radioactivity of added labeled carnitine by the carnitine associated with mitochondria, was usually >90% of that expected from the amount of endogenous total carnitine found initially present in isolated mitochondria ( Fig. 1 and Table III). In a similar experiment with heart mitochondria, where 3 mM external carnitine was present, near equilibrium of exchange was attained in 20 min and at this point the uptake of carnitine, based on radioactivity, corresponded to 103% of the amount of endogenous carnitine initially found in mitochondria.
These data show that almost the entire endogenous carnitine of both liver and heart mitochondria is exchangeable with external carnitine.

Carnitine
Acylcarnitine Translocase and Intramitochondrial Carnitine-Assays of carnitine acylcarnitine translocase activity in mitochondria obtained from livers of rats showed that fasting and alloxan diabetes markedly increased the rates of carnitine transport when expressed as nanomoles of carnitine transported (Table II), but not when expressed as per cent of maximally seen influx (not shown). This suggested that the increase observed in the rates of carnitine exchange could have resulted from an enlargement of carnitine pool in mitochondrial matrix rather than from any increase in the  Table II) that fasting and diabetes not only increased the total carnitine in whole liver, as is known (11,14,15), but also in the mitochondrial fraction. These increases in mitochondrial carnitine did not result from possible increased mitochondrial adsorption of long chain acylcarnitines, concentrations of which are elevated in ketogenic states (33). This is indicated, first, by the finding (Table II) that increases in mitochondrial total carnitine in fasting and alloxan diabetes accompanied parallel increase in the free carnitine content; mitochondrial free carnitine, expressed as per cent of total mitochondrial carnitine, constituted 71% for fed, 67% for fasted, and 68% for alloxan diabetic rats. Secondly, if fasting increases the endogenous intramitochondrial content of carnitine, then in influx experiments despite an apparent leveling off of the net uptake of radioactivity on prolonged incubation (e.g. Fig. lA), the amount of carnitine taken up at such time points, calculated from the specific activity of radioactive carnitine being used, should be greater for mitochondria from livers of fasted as compared to those from fed rats. Table III shows not only that this was observed but that the net uptake of [14C]carnitine approached closely the endogenous total carnitine content of mitochondria.
This indicates that most of the carnitine associated with isolated mitochondria was present in the matrix. The observed increase in the rate of carnitine transport described above could result from elevation of intramitochondrial carnitine if the intramitochondrial carnitine in livers of fed rats normally remains below saturating for the operation of carnitine acylcarnitine translocase. Results of kinetic experiments showed (Table IV) that the apparent K, for carnitine for the carnitine-carnitine exchange of mitochondria from livers of fed rats was 1.8 mM. Fasting had little effect on K,,, but increased the V,,, of carnitine transport (Table IV) in agreement with the other observations described above. Matrix volume of isolated liver mitochondria from fed and fasted rats, determined" as described in Ref. 26, was found to be 1.11 + 0.88 and 1.23 f 0.07 (S.E., n = 5) @mg of protein, respectively. Accordingly, based on the known mitochondrial carnitine content (Table II), the concentration of carnitine in matrix, assuming uniform distribution and accessibility of all carnitine to entire matrix water volume, was calculated to be about 0.19 and 0.29 mM for fed and fasted groups, respectively. tionship between total liver carnitine and mitochondrial carnitine was analyzed further and showed (Fig. 2) that the latter was positively and linearly related to the former under a variety of conditions. A silicone oil technique was worked out for rapid isolation of mitochondria for these analyses and showed higher mitochondrial carnitine content compared to the conventionally isolated mitochondria presumably because losses of carnitine were lower in the former method that required less preparative handling of mitochondria than the conventional isolation procedure. Experiments to determine the mechanism by which intramitochondrial carnitine adjusts to total liver carnitine are currently underway. Effect of Fasting on Carnitine and Deoxycarnitine in Serum and Urine-To elucidate the mechanism(s) responsible for the elevation of liver carnitine, effect of fasting on serum and urinary carnitine was determined. Table  VI shows that l-   (Table VI) on Day 1 and 2 of fasting, respectively, shows that fasting decreased net carnitine synthesis in body by about one-fourth and one-half by 24 and 48 h of fasting. Decreases were also observed for the urinary deoxycarnitine (Table VI) suggesting that fasting lowered deoxycarnitine synthesis as well. The contribution of dietary deoxycarnitine to this decrease was minimal because the rat diet contained little deoxycarnitine (<3% of that of carnitine). Since liver is considered to be the chief site of carnitine biosynthesis in rats (34), it follows from the above that fasting caused a lowering of hepatic carnitine synthesis. However, despite this, an increase in carnitine content of liver, expressed on per g liver weight (Table II), occurred. This suggests, therefore, that fasting caused a slightly increased retention of carnitine in liver. However, Brass and Hoppel (35) have reported that increase in liver carnitine on fasting, expressed on per g of liver wet weight, could be accounted for solely by a conservation of carnitine resulting from a fasting-induced loss of liver weight, relative to body weight, occurring without concurrent losses of hepatic carnitine. Whereas in accord with Brass and Hoppel (35), we find that the data themselves lend to such an interpretation, we believe that it does not represent the actual mechanism involved. A strong suggestive evidence supporting this belief comes from our observation that glucagon infusion markedly (67%) increased liver carnitine (Table  V) without causing any noticeable change in liver weights of rats; the liver weights, expressed on grams per 100 g body weight, were 4.1 f 0.08 for saline (0.9% NaCl solution) infused and 3.9 f 0.09 (n = 6 for both) for glucagon-infused rats. Moreover, we found that as in fasting, glucagon infusionmediated increase in liver carnitine occurred without any significant alteration of serum total carnitine (data not elaborated), indicating that increased extraction of carnitine from serum was probably not the mechanism involved.
The observation that urinary carnitine excretion declined without concurrent decrease in serum carnitine (Table VII) indicates that fasting increased carnitine retention. This is clearly illustrated4 by the fractional excretion rate of carnitine and acylcarnitines, calculated according to glomerular filtration rate of 0.77 ml/min/lOO g body weight (36); fractional excretion of total carnitine thus came to 1.8% for both fed days but decreased to 0.7% on Day 1 of fasting and to 0.4% on Day 2 of fasting. This decrease in the fractional excretion of total carnitine was shared by similar decreases in the fractional excretion of both free as well as of esterified carnitine (data not elaborated).
The possibility that increased mobilization of carnitine from peripheral tissues to liver might explain the increase in liver carnitine on fasting is not supported. Fasting for 48 h did not affect total carnitine in skeletal muscle, heart, and kidney; the total carnitine values of these three tissues of fed and fasted rats were, respectively, as nanomoles per g wet tissue, 1091 f 24 and 1190 + 54 (mean f SE, n = 5 in each group) for skeletal muscle (p > O.l), 1328 & 79 and 1415 -+ 44 (n = 8 in both) for heart (p > 0.2), and 667 f 38 and 555 f 49 (n = 5 in both) for kidney (p > 0.1). The fasting-induced increase of liver carnitine did not result from any increase in the level of the enzyme y-butyrobetaine hydroxylase as the activity of this enzyme, assayed using post-mitochondrial supernatant fraction of liver homogenates, expressed as nanomoles of carnitine formed per min per mg of protein, was 0.25 + 0.011 for fed and 0.24 f 0.008 for fasted (n = 5 in both) group. Table VII shows that although up to 48-h fasting did not affect total serum carnitine, the concentration of free carnitine markedly declined on 24-and 48-h starvation and consequently the ratio of esterified to free carnitine rose at these time points.
This increase in esterified to free carnitine ratio was also seen in urine (Table VI) and is in agreement with the reports (35,37) that appeared while this work was in progress. 4 We thank Dr. Mitchell L. Halperin of the University of Toronto for suggesting this interpretation. We suggest that increased hepatic oxidation of fatty acids such as in fasting leads to the export of acetyl group not only as ketone bodies but also as short chain acylcarnitines to serve as a fuel for extrahepatic tissues just like ketone bodies are known to do. Experiments to ascertain this aspect are in progress. This suggestion is in line with the observations that when hepatic ketogenesis is stimulated by the inclusion of octanoylcarnitine, acetylcarnitine appears in the perfusate (38), and that acetylcarnitine is a physiological substrate for the carnitine-transporting system of heart cells (39). We believe that the results described in this manuscript are consistent with the idea that an enhancement of carnitine-dependent transport of fatty acids into mitochondria occurs under known ketogenic conditions and that this could contribute to the control of ketogenesis. Amatruda et al. (40) have recently reported that the uptake of palmitate by liver mitochondria specially in the presence of carnitine is markedly enhanced by fasting and diabetes. Whereas this effect was considered important for the control of ketone body production, the mechanism of enhanced fatty acid uptake was concluded as being unknown.
Our findings strongly indicate that the enhancement of palmitate uptake in the experiments of Amatruda et al. (40) resulted largely from the enhancement of mitochondrial carnitine acylcarnitine translocase reaction in fasting and diabetes.