Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity <i>in situ</i>

Boon, Mariëtte R.; Zammit, Victor A.

doi:10.1042/bj2490645

Cited by 40 publications

(21 citation statements)

References 38 publications

(58 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Membrane intactness is not invariably achieved by different laboratories, and depends on the method of tissue/cell disruption. By contrast, in whole cells, the preferential permeabilization of the plasma membrane is reproducibly achieved using low concentrations of digitonin, by virtue of the much higher content of cholesterol in this membrane than in the ER (25). The subsequent use of a second membrane-permeabilizing agent, namely alamethacin, a poreforming peptide that acts through an entirely different membrane permeabilization mechanism, allowed the discrete quantification of overt and latent DGAT activities in situ in cells without disruption of the native ER structure.…”

Section: Discussionmentioning

confidence: 99%

“…Ref. 25). Importantly, the additional incubation of cells with alamethacin (20 g/ml), which we used after digitonin to permeabilize the ER, did not increase the amount of glycerol 3-phosphate released by a maximally effective concentration (30 g/ml) of digitonin alone (not shown), indicating that the ability of alamethacin to expose mannose-6-phosphatase activity was entirely due to ER permeabilization and not any additional effect it might have on the integrity of the plasma membrane.…”

Section: Overt and Latent Dgat Activities In Microsomes Andmentioning

confidence: 99%

See 1 more Smart Citation

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Wurie

Buckett

Zammit

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1 ؊/؊ mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.Hypertriglyceridemia is a key biomarker for the metabolic/ insulin resistance syndrome and for associated morbidities, including type-2 diabetes and cardiovascular disease (1). Similarly, excessive accumulation of triglycerides in cytoplasmic lipid droplets results in hepatic steatosis, now recognized as being associated, possibly causatively, with whole body insulin resistance, and which may progress to nonalcoholic fatty liver or steatohepatitis (2, 3). Fasting hypertriglyceridemia is primarily due to the hypersecretion of triglyceride (TAG) 3 by the liver, within very low density lipoproteins (VLDL). Therefore, an understanding of the enzymology involved in triglyceride synthesis, remodeling, storage, and assembly into secreted VLDL is essential for the design of pharmacological strategies aimed at managing dyslipidaemia without the exacerbation of hepatic steatosis, and vice versa.Diacylglycerol acyltransferases (DGATs) catalyze the final reaction of TAG synthesis. Two distinct gene products, DGAT1 and DGAT2, that catalyze most of tissue TAG synthesis have been described (4, 5) but remain relatively poorly characterized, and their respective, nonredundant functions are still to be elucidated. In the present study, we have used two specific inhibitors (which belong to different chemical classes of compounds) of human DGAT1, in combination with the selective permeabilization of the plasma membrane and the ER membrane of whole hepatocytes, to study the sidednes...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Overt and Latent Dgat Activities In Microsomes Andmentioning

confidence: 99%

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Wurie

Buckett

Zammit

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Those studies showed that there was no significant effect of glucagon either on the activity of CPT I in the absence of malonyl-CoA or on the sensitivity of the enzyme to malonyl-CoA inhibition. However, the differences in kinetic characteristics of CPT I induced by starvation and diabetes [3][4][5][6] were entirely preserved, irrespective of the previous treatment of the cells [2]. On the basis of similar experiments, Stephens and Harris [7] concluded that glucagon had a small (25 0%) stimulatory effect on CPT I activity, but that it had no effect on malonyl-CoA sensitivity.…”

Section: Introductionmentioning

confidence: 89%

“…The validity of those studies was questioned [2] on the basis that the use of an anti-CPT II antibody to immunoprecipitate the phosphorylated protein invalidated the conclusion that CPT I was phosphorylated. In an attempt to obtain a functional measure of the effects of glucagon on CPT I in isolated hepatocytes, we conducted studies [2] in which cells were first exposed to glucagon and subsequently permeabilized (in the presence of fluoride and EDTA) with low concentrations of digitonin. This protocol was designed to retain mitochondrial integrity (essential for minimization of the contribution of CPT II towards total CPT activity) as well as any changes in phosphorylation state of CPT I, which was measured in the resulting (washed) cell ghosts.…”

Section: Introductionmentioning

confidence: 99%

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

Guzmán

Kolodziej

Caldwell

et al. 1994

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

The mechanism of activation of mitochondrial overt carnitine palmitoyltransferase (CPT I) by treatment of hepatocytes with okadaic acid (OA) was investigated. Activation was observed when cells were permeabilized with digitonin, but not when a total membrane fraction was obtained by sonication. Both cell disruption methods preserved the activation of phosphorylase observed in OA-treated hepatocytes. Activation of CPT I was also observed in crude homogenates of OA-treated hepatocytes, but it was lost upon subsequent isolation of mitochondria from such homogenates. In all experiments, any activation observed did not depend on the presence or absence of fluoride ions in the permeabilization/homogenization media. When hepatocytes were permeabilized in the absence of fluoride and further incubated with exogenous phosphatases 1 and 2A, the OA-induced activation of CPT was not reversed, whereas the activation of glycogen phosphorylase in the same cells was rapidly reversed. Treatment of hepatocytes with OA, followed by permeabilization and incubation before assay of CPT I, demonstrated that OA had no short-term effect on the sensitivity of CPT I to malonyl-CoA, although the difference in sensitivity between cells isolated from fed and starved rats was fully preserved. Incubation of isolated mitochondria or purified mitochondrial outer membranes with cyclic AMP-dependent or AMP-activated protein kinases, under phosphorylating conditions, did not affect the activity of CPT I or its sensitivity to malonyl-CoA inhibition. Under the same conditions, the use of [32P]ATP resulted in the labelling of several outer-membrane proteins but, unlike [3H]etomoxir-labelled CPT I, none of them was specifically removed from membrane extracts by a specific polyclonal antibody to the enzyme. We conclude that the increase in overt CPT activity observed in permeabilized hepatocytes is not due to direct phosphorylation of CPT I, but may involve interactions between the mitochondrial outer membrane and other membranous or soluble cytosolic components of the cell.

show abstract

“…Although carnitine palmitoyl transferase I is also considered an important control of the ketogenic flux in liver, under certain conditions control shifts away from this enzyme to an intramitochondrial control site (3,4). Many groups suggest that this control lies within the pathway from acetyl-CoA to ketonic bodies, the HMG-CoA pathway (5,6).…”

mentioning

confidence: 99%

Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes.

Ayté

Gil‐Gómez

Haro

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass. Identity of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase. (i) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase. (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity. (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme. The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria. These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA and CoA. In liver this reaction occurs in two essentially different pathways: (i) the production of mevalonate from HMG-CoA prior to the synthesis of sterols and isoprenoids of various functions; and (ii) the conversion of HMG-CoA into acetoacetate and then into other related compounds by means of the ketogenetic pathway that occurs in mitochondria.Mitochondrial HMG-CoA synthase has been assumed to be the rate-limiting enzyme of ketogenesis in rat liver (1, 2). Although carnitine palmitoyl transferase I is also considered an important control of the ketogenic flux in liver, under certain conditions control shifts away from this enzyme to an intramitochondrial control site (3, 4). Many groups suggest that this control lies within the pathway from acetyl-CoA to ketonic bodies, the HMG-CoA pathway (5, 6).Gil et al. (7)(8)(9) speculated that both cytosolic and mitochondrial HMG-CoA synthase enzymes were products of the same gene, transcribed through an alternative splicing mechanism. This hypothesis was generated from the observation that the catalytic domain of both synthases was nearly homologous (7, 10) and that an alternative splicing pattern of exon 2 in cytosolic HMG-CoA synthase occurs among various tissues through development. This also holds true between human and hamster genes (8, 9).However, because mitochondrial and cytoplasmic HMGCoA synthases differ in charge and immunological properties (11) and no clear splicing...

show abstract

Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ

Cited by 40 publications

References 38 publications

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

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

Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes.

Contact Info

Product

Resources

About