Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes

Guzmán, Manuel; Kolodziej, M P; Caldwell, A M; Corstorphine, C G; Zammit, Victor A.

doi:10.1042/bj3000693

Cited by 33 publications

(35 citation statements)

References 25 publications

(52 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Assay of CPT-I Activity in Permeabilized Hepatocytes-After incubation of the hepatocytes with the additions indicated in each case, CPT-I activity was determined in digitonin-permeabilized hepatocytes as the tetradecylglycidate-sensitive incorporation of radiolabeled L-carnitine into palmitoylcarnitine (9,10). In the "one-step assay" (Table II), cell permeabilization and assay of enzyme activity are simultaneously performed (9).…”

Section: Materials-l-[methyl-mentioning

confidence: 99%

“…Assay of CPT-I Activity in Isolated Mitochondria-Mitochondria were isolated either from hepatocytes or from intact liver and CPT-I activity was measured as the malonyl-CoA-sensitive incorporation of radiolabeled L-carnitine into palmitoylcarnitine exactly as described before (10). When CPT-I activity was determined in suspensions of mitochondria containing cytoskeletal fractions (Figs.…”

Section: Materials-l-[methyl-mentioning

confidence: 99%

“…In this "two-step" assay, hepatocytes are permeabilized with digitonin, and then extensively washed with a medium containing 10 mM Tris-HCl (pH 7.4), 50 mM potassium fluoride, 100 mM KCl, 2.5 mM EDTA, and 2.5 mM EGTA. The permeabilized cell pellets were taken up in that medium with the additions indicated and CPT-I activity was subsequently determined after preincubation at 37°C for 5 min (10).…”

Section: Materials-l-[methyl-mentioning

confidence: 99%

“…3 H]Carnitine, carrier-free [ 32 P]P i , [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] acetyl-CoA, the ECL detection kit, and the protein kinase C assay kit were from Amersham International (Amersham, Bucks, United Kingdom). Tetradecylglycidate was kindly donated by Dr. J. M. Lowenstein (Brandeis University, Waltham, MA).…”

Section: Materials-l-[methyl-mentioning

confidence: 99%

“…Studies using permeabilized hepatocytes have shown that various agents exert short-term changes in CPT-I activity in parallel with changes in the rate of long-chain fatty acid oxidation (3,9). These short-term changes in hepatic CPT-I activity are assumed to be mediated by a malonyl-CoA-independent mechanism, since they survive cell permabilization, extensive washing of the permeabilized cells (to allow complete removal of malonyl-CoA), and subsequent preincubation of the cell ghosts at 37°C before determination of CPT-I activity (to allow recovery of the original conformational state of CPT-I) (10). Evidence has also been presented showing that the stimulation of hepatic CPT-I by the phosphatase inhibitor okadaic acid (OA), used as a model compound to study the short-term regulation of CPT-I, does not involve the direct phosphorylation of CPT-I (10).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity

Velasco¹,

Geelen²,

Pulgar³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

The mechanism of malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I (CPT-I) activity was investigated. In a first series of experiments, the possible involvement of the cytoskeleton in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment of permeabilized hepatocytes with trypsin produced around 50% stimulation of CPT-I activity. This effect was absent in cells that had been pretreated with okadaic acid (OA) and seemed to be due to the action of trypsin on cell component(s) distinct from CPT-I. (ii) Incubation of intact hepatocytes with 3,3-iminodipropionitrile, a disruptor of intermediate filaments, increased CPT-I activity in a non-additive manner with respect to OA. Taxol, a stabilizer of the cytoskeleton, prevented the OA-and 3,3-iminodipropionitrile-induced stimulation of CPT-I. (iii) CPT-I activity in isolated mitochondria was depressed in a dose-dependent fashion by the addition of a total cytoskeleton fraction and a cytokeratin-enriched cytoskeletal fraction, the latter being 3 times more potent than the former. In a second series of experiments, the possible link between Ca 2؉ /calmodulin-dependent protein kinase II (Ca 2؉ /CM-PKII) and the cytoskeleton was studied in the context of CPT-I regulation. The data of these experiments indicate that (i) purified Ca 2؉ /CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolated mitochondria, (ii) purified Ca 2؉ /CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratin-enriched fraction, and (iii) the Ca 2؉ /CM-PKII inhibitor KN-62 prevented the OA-induced phosphorylation of cytokeratins in intact hepatocytes. Results thus support a novel mechanism of short-term control of hepatic CPT-I activity which may rely on the cascade Ca 2؉ /CM-PKII activation 3 cytokeratin phosphorylation 3 CPT-I de-inhibition.Mitochondrial fatty acid oxidation in liver provides a major source of energy to this organ and supplies extrahepatic tissues with ketone bodies as a glucose-replacing fuel (1, 2). Carnitine palmitoyltransferase I (CPT-I), 1 the carnitine palmitoyltransferase of the mitochondrial outer membrane, catalyzes the pace-setting step of long-chain fatty acid translocation into the mitochondrial matrix (1-5). Moreover, recent determination of flux control coefficients of the enzymes involved in hepatic long-chain fatty acid oxidation shows that CPT-I plays a pivotal role in controlling the flux through this pathway under different substrate concentrations and pathophysiological states (6, 7). CPT-I is subject to long-term regulation in response to alterations in the nutritional and hormonal status of the animal (1, 2, 5). Short-term control of CPT-I activity involves inhibition by malonyl-CoA, the product of the reaction catalyzed by acetyl-CoA carboxylase (8). Since the latter enzyme is a key regulatory site of fatty acid synthesis de novo (cf. Refs. 1-5), malonyl-CoA inhibition of CPT-I allows an elegant explanati...

show abstract

Section: Materials-l-[methyl-mentioning

confidence: 99%

Section: Materials-l-[methyl-mentioning

confidence: 99%

Section: Materials-l-[methyl-mentioning

confidence: 99%

Section: Materials-l-[methyl-mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity

Velasco¹,

Geelen²,

Pulgar³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system

et al. 2000

View full text Add to dashboard Cite

An efficient regulation of fuel metabolism in response to internal and environmental stimuli is a vital task that requires an intact carnitine system. The carnitine system, comprehensive of carnitine, its derivatives, and proteins involved in its transformation and transport, is indispensable for glucose and lipid metabolism in cells. Two major functions have been identified for the carnitine system: (1) to facilitate entry of long-chain fatty acids into mitochondria for their utilization in energy-generating processes; (2) to facilitate removal from mitochondria of short-chain and medium-chain fatty acids that accumulate as a result of normal and abnormal metabolism. In cancer patients, abnormalities of tumor tissue as well as nontumor tissue metabolism have been observed. Such abnormalities are supposed to contribute to deterioration of clinical status of patients, or might induce cancerogenesis by themselves. The carnitine system appears abnormally expressed both in tumor tissue, in such a way as to greatly reduce fatty acid beta-oxidation, and in nontumor tissue. In this view, the study of the carnitine system represents a tool to understand the molecular basis underlying the metabolism in normal and cancer cells. Some important anticancer drugs contribute to dysfunction of the carnitine system in nontumor tissues, which is reversed by carnitine treatment, without affecting anticancer therapeutic efficacy. In conclusion, a more complex approach to mechanisms that underlie tumor growth, which takes into account the altered metabolic pathways in cancer disease, could represent a challenge for the future of cancer research.

show abstract

Regulation of Ketogenesis in Liver

Zammit

2001

Comprehensive Physiology

View full text Add to dashboard Cite

The sections in this article are: Roles of Insulin and Glucagon in Substrate Supply for Hepatic Ketogenesis Effects of Ketones on the Endocrine Pancreas and Adipose Tissue Intrahepatic Partitioning of Fatty Acids Between Oxidation and Esterification Pancreatic Hormone Effects on Enzyme Substrate Availability Expression of Enzyme Activity Role of Malonyl CoA Role of Carnitine Palmitoyltransferase I Membrane Topology and Membrane‐Protein Interactions Control of Carnitine Palmitoyltransferase I over Ketogenesis: Effects of Altered Insulin Status In Vivo Monitoring of Hepatic Acyl‐CoA Partitioning Intramitochondrial Regulation of Ketogenesis Modulation of Mitochondrial 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase Activity Examples of Physiological Conditions Involving Pancreatic Hormone Regulation of Ketogenesis General Comments

show abstract

Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes

Cited by 33 publications

References 25 publications

Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity

Malonyl-CoA-independent Acute Control of Hepatic Carnitine Palmitoyltransferase I Activity

Cancer and anticancer therapy-induced modifications on metabolism mediated by carnitine system

Regulation of Ketogenesis in Liver

Contact Info

Product

Resources

About