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65 Bach, I. et al. (1997) A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev. 11, 1370–1380 66 Lipkin, S.M. et al. (1993) Identification of a novel zinc finger protein binding a conserved element critical for Pit-1-dependent growth hormone gene expression. Genes Dev. 7, 1674–1687 67 Topilko, P. et al. (1997) Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. Mol. Endocrinol. 12, 107–122 68 Chen, S. et al. (1999) Growth hormone deficiency with ectopic neurohypophysis: anatomical variations and relationship between the visibility of the pituitary stalk asserted by magnetic resonance imaging and anterior pituitary function. J. Clin. Endocrinol. Metab. 84, 2408–2413 69 Jenkins, J.S. et al. (1976) Hypothalamic– pituitary function in patients with craniopharyngiomas. J. Clin. Endocrinol. Metab. 43, 394 70 Clayton, P.E. et al. (1999) Signal transduction defects in growth hormone insensitivity. Acta Paediatr. Suppl. 88, 174–178; discussion 179 71 Barreca, A. et al. (1998) Short stature associated with high circulating insulin-like

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growth factor (IGF)-binding protein-1 and low circulating IGF-II: effect of growth hormone therapy. J. Clin. Endocrinol. Metab. 83, 3534–3541 Rosenfeld, R.G. et al. (1994) Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr. Rev. 15, 369–390 Burren, C.P. et al. (1999) Serum levels of insulin-like growth factor binding proteins in Ecuadorean children with growth hormone insensitivity. Acta Paediatr. Suppl. 428, 185–191 Liu, J-P. et al. (1993) Mice carrying null mutations to the genes encoding insulinlike growth factor I (IGF-I) and type 1 IGF receptor (IGF1R). Cell 75, 59–72 Jain, S. et al. (1998) Insulin-like growth factor-I resistance. Endocr. Rev. 19, 625–646 Godfrey, P. et al. (1993) GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor funtion. Nat. Genet. 4, 227–232 Lin, S-C. et al. (1993) Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature 364, 208–213 Takeuchi, T. et al. (1990) Molecular mechanism of growth hormone (GH) deficiency in the spontaneous dwarf rat: detection of abnormal splicing of GH messenger

Do Cytoskeletal Components Control Fatty Acid Translocation into Liver Mitochondria? Manuel Guzmán, Guillermo Velasco and Math J.H. Geelen

For two decades it has been assumed that inhibition of carnitine palmitoyltransferase I (CPT-I) by malonyl-CoA represents the main regulatory mechanism of liver ketogenesis. However, recent evidence indicates that CPT-I activity is also controlled by interactions between mitochondria and cytoskeletal components. This newly recognized mechanism emphasizes the emerging role of the cytoskeleton in the regulation of metabolic pathways. Mitochondrial fatty acid oxidation in liver is a major source of energy for this organ and supplies extrahepatic tissues (including the brain) with ketone bodies when glucose is becoming scarce as M. Guzmán and G. Velasco are at the Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040-Madrid, Spain. M.J.H. Geelen is at the Laboratory of Veterinary Biochemistry, Utrecht University, PO Box 80.176, 3508 TD Utrecht, The Netherlands.

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fuel1. Hepatic ketogenesis is enhanced in physiological conditions such as starvation and in some mammalian species in the suckling period2. In addition, the uncontrolled production of ketone bodies, leading to ketosis, is a metabolic aberration that is important both in human (e.g. diabetes) and veterinary medicine (e.g. pregnancy toxaemia in cattle and sheep). A large number of classic studies, corroborated by more recent analyses of metabolic control,

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ribonucleic acid by the polymerase chain reaction. Endocrinology 126, 31–38 Zhou, Y. et al. (1997) A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc. Natl. Acad. Sci. U. S. A. 94, 13215–13220 Amselem, S. et al. (1989) Laron dwarfism and mutations of the growth hormonereceptor gene. New Engl. J. Med. 32, 989–995 Godowski, P.J. et al. (1989) Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Larontype dwarfism. Proc. Natl. Acad. Sci. U. S. A. 86, 8083–8087 Li, H. et al. (1994) Gsh-4 encodes a LIMtype homeodomain, is expressed in the developing central nervous system and is required for early postnatal survival. EMBO J. 13, 2876–2885 Parganas, E. et al. (1998) Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93, 385–395 Udy, G.B. et al. (1997) Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc. Natl. Acad. Sci. U. S. A. 94, 7239–7244

have established that the enzyme carnitine palmitoyltransferase I (CPT-I) plays a central role in the control of hepatic ketogenesis under different experimental conditions and pathophysiological states1,3. CPT-I is located at the mitochondrial outer membrane and is part of the carnitine-dependent machinery of long-chain fatty acid translocation into mitochondria (Fig. 1). In the mitochondrial matrix, fatty acyl-CoA is rapidly catabolized to acetyl-CoA, yielding ketone bodies when the rate of boxidation exceeds the capacity of the citric acid cycle. CPT-I is potently inhibited by malonyl-CoA, which, in turn, is the product of the reaction catalyzed by acetyl-CoA carboxylase (ACC), a key regulatory enzyme of fatty acid synthesis. Since the pioneering work of McGarry et al.4 in the 1970s, evidence has accumulated highlighting the physiological and clinical importance of malonyl-CoA in the coordinate control of fatty acid synthesis and oxidation3. • Evidence for the Existence of a Malonyl-CoA-independent Regulation of CPT-I During the 1980s, malonyl-CoA was considered to be the sole modulator of

1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(99)00223-4

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Fatty acids

Acyl-CoA

CoA-SH

ACS

CPT-I

Carnitine

Hence, we started to search for those components.

Malonyl-CoA

MOM

Acylcarnitine

MIM

T CPT-II Carnitine

Acylcarnitine Acyl-CoA CoA-SH β-Oxidation Carnitine trends in Endocrinology and Metabolism

Figure 1. Carnitine-dependent translocation of fatty acids into the mitochondrial matrix. Fatty acids are activated to their CoA esters by an ACS in the MOM. CPT-I catalyses the synthesis of an acylcarnitine complex from acyl-CoA and carnitine. The acylcarnitine is subsequently transferred to the MIM, in which the consecutive action of T and CPT-II translocates the acyl-CoA moiety to the mitochondrial matrix for oxidative metabolism. CPT-I is inhibited by malonyl-CoA at a site in the outer leaflet of the MOM. Abbreviations: ACS, acyl-CoA synthetase; CoA-SH, co-enzyme A; CPT-I/II, carnitine palmitoyltransferase I/II; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; T, carnitine:acylcarnitine translocase.

CPT-I activity. By that time, it had been proposed that hepatic CPT-I activity could be controlled by phosphorylation–dephosphorylation. However, several observations argued strongly against that possibility. For example, incubation of isolated mitochondria or purified mitochondrial outer membranes with various protein kinases and phosphatases did not affect CPT-I activity. Moreover, after incubation of isolated hepatocytes with different hormones, followed by the isolation of mitochondria and assay of CPT-I activity, no changes in CPT-I activity were found. Although discouraging, these observations prompted us to pose the following question: might this lack of stable changes of CPT-I activity be artifacticious? In other words, we postulated that this type of modulation of CPT-I was lost upon isolation of mitochondria. To test that hypothesis, we developed a procedure for measuring CPT-I activity in permeabilized hepatocytes. Permeabilizing the plasma membrane enables intracellular enzymes to be investigated in a more or less natural

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environment, and alleviates the necessity of preparing cellular fractions for enzyme assay. The use of such methods, both in their original form and in further improved modifications, revealed that short-term incubations of hepatocytes with a number of cellular effectors did change CPT-I activity coordinately with the rate of ketogenesis. Furthermore, we showed that small cytosolic molecules such as malonylCoA leaked completely out of the permeabilized hepatocytes, demonstrating that a malonyl-CoA-independent mechanism was responsible for the observed changes in CPT-I activity5,6. This notion was strengthened by experiments with cellular effectors (such as vanadate, a protein tyrosine phosphatase inhibitor, and extracellular ATP, a Ca21-mobilizing agent) that modulate hepatic CPT-I activity in a direction opposite to that expected from their respective effects on intracellular malonyl-CoA levels5. Moreover, other observations also suggested that both non-diffusible and diffusible cell components were involved in the malonyl-CoA-independent regulation of CPT-I (Ref. 6).

• On the Nature of the MalonylCoA-independent Regulation of CPT-I Our earlier observation that the cytoskeletal stabilizer taxol prevented the malonyl-CoA-independent changes of CPT-I activity induced by various agents7 focused our attention on the cytoskeleton as a potential non-diffusible regulator of CPT-I activity. This hypothesis was attractive in view of the emerging role of the cytoskeleton in the regulation of metabolic pathways, signal transduction systems and mitochondrial dynamics8,9. In addition, the observation that the bulk of the CPT-I protein faces the cytoplasmic side of the mitochondrial outer membrane10 underscored the possibility of interactions between CPT-I and the cytoskeleton. Subsequent experiments using specific modulators of cytoskeletal dynamics, as well as reconstitution experiments with purified mitochondrial and cytoskeletal fractions, showed that CPT-I activity is indeed modulated by the aggregation state of the cytoskeleton. In particular, depolymerization of the cytoskeleton, induced either by phosphorylation of cytoskeletal components or by pharmacological disruption, freed CPT-I from inhibitory constrictions by the cytoskeleton. Among the different components of the cytoskeleton, intermediate filaments and, especially, cytokeratins 8 and 18, were the most likely candidates for modulation of CPT-I activity in hepatocytes11. One of the tools used to study the malonyl-CoA-independent regulation of CPT-I is okadaic acid. Incubation of hepatocytes with this potent inhibitor of protein phosphatases 1 and 2A resulted in a remarkable stimulation of CPT-I (Ref. 5). It was unequivocally shown that this effect of okadaic acid was independent of malonyl-CoA and not linked to a direct phosphorylation of CPT-I (Ref. 6). Surprisingly, addition of purified protein phosphatases 1 and 2A to cell ghosts did not produce any effect on CPT-I activity. A tentative interpretation of this result was that a TEM Vol. 11, No. 2, 2000

cellular component, activated by phosphorylation and released from the cells upon permeabilization, was necessary to affect the aggregation state of the cytoskeleton and therefore CPT-I activity. The use of specific inhibitors of protein kinases and phosphatases, in concert with reconstitution experiments and studies on cytokeratin phosphorylation in intact hepatocytes, collectively pointed to Ca21–calmodulin-dependent protein kinase II (Ca21–CM-PKII) as candidate molecule11,12. This kinase is activated by autophosphorylation of key residues within its autoinhibitory domain. Therefore, incubation of intact hepatocytes with okadaic acid might well result in the phosphorylation and subsequent activation of Ca21–CMPKII (Refs 12,13). Interestingly, Ca21– CM-PKII is known to play a major role both in the phosphorylation and functional integrity of liver cytokeratins in vivo13 and in the okadaic acid-induced disruption of cytoskeletal structures in the liver cell14. A clear link between Ca21–CM-PKII activation, intermediate filament disruption and CPT-I activation was provided by the observation that purified Ca21–CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratinenriched fraction11. Is Ca21–CM-PKII the sole kinase responsible for the malonyl-CoA-independent control of CPT-I activity? The search for other kinases focused on the AMP-activated protein kinase (AMPK) because of its putative regulatory role in the mainstream of hepatic lipid metabolism15. As expected, pharmacological activation of AMPK inhibited ACC, decreased malonyl-CoA levels and thus stimulated CPT-I in hepatocytes. However, further analysis demonstrated that the AMPK-induced activation of hepatic CPT-I relied partially on a malonyl-CoA-independent, cytoskeleton-dependent mechanism16. In fact, subsequent experiments corroborated the hypothesis that AMPK might control the activity of liver CPT-I by a mechanism similar to that outlined above for Ca21–CM-PKII (Ref. 17). All these data led us to propose a model for the malonyl-CoA-independent control of hepatic CPT-I activity TEM Vol. 11, No. 2, 2000

Ca2+–CM-PKII (Auto) kinase

AMPK

P

Pases 1/2A

AMPKK P

Pas 2C

AMPK

Ca2+–CM-PKII

Phosphorylation of cytoskeletal components

CPT-I

Ketogenesis trends in Endocrinology and Metaboli

Figure 2. Proposed model for the malonyl-CoA-independent control of hepatic CPT-I activity. Activation of Ca21–CM-PKII by an (auto)kinase or AMPK by AMPKK induces the phosphorylation of cytoskeletal components, most likely cytokeratins 8 and 18, thereby disrupting intermediate filaments. Thus, inhibitory interactions between the cytoskeleton and mitochondrial components are lost, making CPT-I and ketogenesis more active. Abbreviations: AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase; Ca21–CM-PKII, Ca21–calmodulin-dependent protein kinase II; CPT-I, carnitine palmitoyltransferase I; Pase, protein phosphatase.

(Fig. 2). Activation of Ca21–CM-PKII or AMPK induces the phosphorylation of cytokeratins 8 and 18, thereby disrupting intermediate filaments. As a consequence, inhibitory interactions between the cytoskeleton and mitochondrial components are lost, rendering CPT-I and ketogenesis more active. The identity of the mitochondrial components interacting with the cytoskeleton is as yet unknown. In addition, it cannot be excluded that other protein kinases are also involved. • Malonyl-CoA-independent Regulation of CPT-I in a Physiological Context It should be stated clearly that the notion of control of fatty acid translocation into mitochondria by modulation of the interactions between CPT-I and cytoskeletal components (that is, by a malonyl-CoA-independent mechanism) does not contradict the importance of malonyl-CoA as a physiological modulator of CPT-I. Most likely, the malonyl-CoA-dependent and malonylCoA-independent control of hepatic CPT-I activity operate in concert, and the latter mechanism might effectively tune the former one. This should certainly occur in the case of Ca21–CMPKII and AMPK, because both kinases are able to decrease malonyl-CoA levels

by phosphorylating and inactivating ACC, and to disrupt intermediate filaments by phosphorylating cytokeratins 8 and 18. In fact, evidence has been presented showing that in intact hepatocytes the stimulation of CPT-I by AMPK is a result of the coordinate action of both mechanisms16. Another link between the two mechanisms of control of CPT-I activity is that a small proportion of hepatic ACC appears to be attached to the cytoskeleton18. This binding of ACC was shown to be dependent on the aggregation state of the cytoskeleton, and its disruption induced by okadaic acid led to the detachment of ACC. Interestingly, the Ca21–CM-PKII inhibitor KN-62 prevented both the okadaic acid-induced removal of ACC from the cytoskeleton and the okadaic acid-induced inhibition of ACC (Ref. 18). Furthermore, the idea has been put forward that one of the two ACC isoforms expressed by the liver (the 280-kDa isoform) might interact with the outer leaflet of the mitochondrial outer membrane to channel malonyl-CoA for CPT-I inhibition19. It is clear that physical interaction between ACC (the source of malonylCoA), CPT-I (the target of malonyl-CoA) and the cytoskeleton (the common support of both enzymes) would direct the concerted performance of the 51

Ceramide

Ceramide

APOPTOSIS Palmitate

Cytoskeletal components

APOPTOSIS

Cell transformation BCL-2

Palmitate

P

Okadaic acid ? CPT-I

P

P BCL-2

P ? CPT-I

Ketone bodies trends in Endocrinology and Metabolism

Figure 3. Proposed model for the role of CPT-I in the onset of apoptosis. The high CPT-I activity seen in either hepatoma cells compared with hepatocytes or in hepatocytes exposed to the protein phosphatase inhibitor okadaic acid, is caused by the loss of inhibitory interactions between mitochondria and cytoskeletal components. This enhanced CPT-I activity favours the mitochondrial metabolism of fatty acids, which are precursors of de novo ceramide synthesis, thereby allowing transformed cells to withstand apoptosis31. It is still unknown whether BCL-2 modulates CPT-I activity. Abbreviation: CPT-I, carnitine palmitoyltransferase I.

malonyl-CoA-dependent and malonylCoA-independent limbs of CPT-I regulation. We favour a model in which CPTI activity within the cell depends on: (1) ACC activity and/or malonyl-CoA levels; (2) the constrictions imposed on CPT-I by the cytoskeleton; and (3) the intracellular localization of ACC. The insulin to glucagon ratio of plasma is considered the most important factor controlling liver lipid metabolism in vivo20. Accordingly, both insulin and glucagon induce changes in the activity and phosphorylation extent of liver ACC (Ref. 21), as well as changes in CPT-I activity that parallel changes in ketogenesis and that are retained in cell ghosts4. Several protein kinases have been shown to phosphorylate ACC in vitro; however, there is ample evidence demonstrating that in intact hepatocytes and in the liver in vivo AMPK is the major kinase responsible for the inactivation of ACC by phosphorylation15. Insulin simultaneously inhibits AMPK and activates ACC in hepatocytes, but no signal transduction mechanism has been ascribed so far15,22. • Pathological Implications of the Malonyl-CoA-independent Regulation of CPT-I The existence of the malonyl-CoAindependent regulation of CPT-I predicts that CPT-I activity, as affected by

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intermediate filaments, might change under pathological situations in which the organization of the cytoskeleton is altered. Traditionally, intermediate filaments have been considered static components of the cytoskeleton – a notion that has completely changed during the past few years. For example, the organization of intermediate filaments, as well as the phosphorylation pattern of cytokeratins 8 and 18, change significantly in a number of pathological changes of the liver and in other extreme situations such as transformation, mitosis, apoptosis and stress23,24. For this reason, we tested whether the regulatory properties of CPT-I were altered in hepatoma cells25. We found that CPT-I-specific activity was similar in mitochondria isolated from hepatoma cells and from normal hepatocytes, whereas that of permeabilized hepatocytes was about half of that of permeabilized hepatoma cells. In addition, CPT-I activity in hepatoma cells was insensitive to okadaic acid, whereas in hepatocytes this compound increased CPT-I activity about twofold. Hence, the high CPT-I activity present in hepatoma cells might be reached by hepatocytes upon challenge by okadaic acid. Moreover, reconstitution experiments showed that the cytoskeleton of hepatocytes caused a more powerful inhibition of CPT-I than did the

cytoskeleton of hepatoma cells. A plausible interpretation is that in hepatocytes okadaic acid liberates CPT-I from certain constrictions imposed by cytoskeletal components that are not present in isolated mitochondria or in transformed liver cells. In short, treatment of hepatocytes with okadaic acid, a well-known tumour promoter, gives rise to a ‘regulatory phenotype’ of CPTI, which is similar to that displayed by hepatoma cells. A connection between CPT-I and apoptosis has been postulated recently (Fig. 3). Palmitoyl-CoA, a substrate for CPT-I, is also a preferred substrate for ceramide synthesis de novo. Paumen et al.26 have observed that pharmacological inhibition of CPT-I results in: (1) an accumulation of palmitate in the cytoplasm; (2) an increase of ceramide synthesis; and (3) apoptosis. Therefore, cells expressing a high CPT-I activity should be more resistant to palmitateinduced apoptosis – which is the case for hepatoma cells25,26. The observations that CPT-I interacts directly with the anti-apoptotic protein BCL-2 in the mitochondrial outer membrane27, and that CPT-I itself is a ceramide-activated enzyme28, make the story even more intriguing. The activity of the BCL-2 protein family might be controlled by extracellular signals through protein kinase TEM Vol. 11, No. 2, 2000

B/Akt. This kinase phosphorylates the pro-apoptotic protein BAD, which is then sequestered by a cytosolic 14-3-3 protein. This prevents BAD from binding to and inhibiting BCL-2/BCL-xL (Ref. 29). During mitosis, or after cell treatment with okadaic acid, cytokeratins 8 and 18 become hyperphosphorylated, and the latter bind to 14-3-3 proteins30. Although it is still unknown how the interactions between CPT-I and BCL-2, BCL-2 and BAD, and cytokeratins and 14-3-3 proteins affect CPT-I activity, a speculative but attractive model emerges in which a survival/mitosis signal would lead to the activation of protein kinase B/Akt, the phosphorylation of BAD and cytokeratins and their binding to 14-3-3 proteins. These two events would lead to the de-inhibition of BCL2 and CPT-I, thereby suppressing apoptosis. The possibility that the antiapoptotic effect of BCL-2 might partially depend on the activity of CPT-I is an interesting topic for future research. In view of the well-established role of mitochondria in the onset of apoptosis31, the above data collectively suggest a more general role for CPT-I in the regulation of apoptosis. • Concluding Remarks Inhibition of CPT-I by malonyl-CoA has been recognized during the past two decades as the key regulatory event of fatty acid oxidation. More recent data show that CPT-I activity might also be controlled by interactions between mitochondria and the cytoskeleton. This might have important implications in the overall physiology of the cell, particularly in the balance between apoptosis and survival. According to modern metabolic concepts, not only the activity and levels, but also the organization and topology of enzymes/substrates are subject to fine control within the cell. The existence of sequential complexes of enzymes (metabolons) and of ordered transfer of intermediates among enzymes (substrate channelling) is well established. The precise regulation of many of these events might depend on the dynamics of the cytoskeleton. We eagerly anticipate the answers to a number of exciting TEM Vol. 11, No. 2, 2000

questions on the regulatory role of the cytoskeleton in crucial processes such as metabolic regulation, cell growth and apoptosis.

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Acknowledgements Research in our laboratories is supported by Spanish Comisión Interministerial de Ciencia y Tecnología (grant SAF96/0113), Fondo de Investigación Sanitaria (grant FIS 97/0039) and by the Netherlands Foundation for Chemical Research (SON), with financial aid from the Netherlands Organization for Scientific Research (NWO).

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