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Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C

Université de Bourgogne, UPRES Lipides et Nutrition EA 2422, Faculté des Sciences, F-21000 Dijon, France; and
^* Laboratoire de Pharmacologie Moléculaire, Faculté de Pharmacie, Université de Rennes I, France

¹Correspondence: Université de Bourgogne, UPRES Lipides et Nutrition EA 2422, Faculté des Sciences, 6 Blvd. Gabriel, F-21000 Dijon, France. E-mail: naim.khan{at}u-bourgogne.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We synthesized diacylglycerols (DAGs) containing -6 or -3 polyunsaturated fatty acids [i.e., 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG)] and assessed their efficiency on activation of conventional (, ßI, ) and novel (, ) protein kinase C (PKC). SAG exerted significantly higher stimulatory effects than SDG and SEG on activation of PKC and PKC. Activation of PKCßI by SEG and SDG was higher than that by SAG. Activation of PKC did not differ significantly among DAG molecular species. Addition of SAG to assays containing SEG and SDG exerted additive effects on activation of and , but not on ßI and , isoforms of PKC. SDG- and SEG-induced activation of PKC was significantly curtailed by the addition of SAG. Three DAG species significantly curtailed the PMA-induced activation of ßI, , and , but not of and , isoforms of PKC. Our study demonstrates for the first time that in vitro activation of different PKC isoenzymes vary in response to different DAG species, and one can envisage that this differential regulation may be responsible for their in vivo effects on target organs.—Madani, S., Hichami, A., Legrand, A., Belleville, J., Khan, N. A. Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C.

Key Words: DAG • -3/-6 fatty acids • PKC isoenzymes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE PROTEIN KINASE C (PKC) has been implicated as a pivotal regulatory element in a variety of cellular functions such as gene expression, growth, differentiation, and exocytosis (1) . This enzyme has also been implicated in phosphorylation of several neuronal proteins that regulate release of neurotransmitters implicated in long-term potentiation (1) . Eleven isoenzymes of PKC have been identified and classified into three subclasses: the Ca²⁺-dependent or conventional PKCs (, ßI, ßII, and ), the Ca²⁺-independent or novel PKCs (, , , and ), and the Ca²⁺- and diacylglycerol (DAG) -independent or atypical PKCs (, , and ) (1) . PKC, ßI, ßII, , , and isoforms seem to be ubiquitously distributed in brain, lung, spleen, thymus, and skin whereas PKC is exclusively found in the brain and spinal cord (1) . As lipids are required for PKC activation, the activated enzyme is exclusively membrane-bound where it phosphorylates the target proteins.

Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) is thought to require DAG as an activator and phosphatidylserine (PS) as a cofactor of activation. The presence of DAG induces a selective increase in affinity of cPKCs and nPKCs for PS (2) . This high-affinity interaction of DAG has been found to be stronger for phosphatidyl-L-serine compared with other acidic lipids such as phosphatidyl-D-serine (3) . During cell activation via membrane receptors, DAG is produced in a biphasic way. The transient production of DAG is assured by the hydrolysis of phosphatidylinositol 4,5 biphosphate (PIP₂) catalyzed by phospholipase C. The second sustained phase of DAG generation is associated with an increase in the activation of phospholipase D-catalyzed phosphatidylcholine (PC) hydrolysis, producing phosphatidate that can be converted to DAG by the action of phosphatidate phosphohydrolase (4 , 5) .

The activation of PKC by DAG is stereospecific and therefore requires sn-1,2-DAG. On the other hand, sn-1,3-DAG and sn-2,3-DAG are not effective in the activation of PKC (6) . The presence of polyunsaturated fatty acids (PUFAs) on sn-2 position of DAG also seems to be important to activate PKC. For instance, in porcine aortic cells, DAG generated from PIP₂ hydrolysis activates PKC whereas that produced from PC is not able to activate this enzyme because PC is usually less unsaturated than PIP₂ (7) .

The anti-inflammatory, antithrombotic. and immunomodulatory effects of diets supplemented with -3 PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been widely studied for several years (8) . A plausible hypothesis is that these highly unsaturated fatty acids replace and reduce cellular membrane contents of arachidonic acid (AA), an -6 PUFA, to exert the effects through production of substantially less active eicosanoids and a decrease in the production of cytokines such as tumor necrosis factors and interleukin 1 (9) . Hence, the -3 PUFAs incorporated into plasma membrane phospholipids could affect PKC activity either directly or via DAG production after phospholipid hydrolysis. Several studies have demonstrated that, as free fatty acids, AA, EPA, and DHA are equally able to activate PKC in rat brain (10) , colon (11) , and other cell systems (12 , 13) .

Not many studies are available on the role of different DAG species (containing exogenous PUFAs) in the activation of PKC. Marignani et al. (14) have shown for the first time that 1-setaroyl, 2-arachidonoyl-glycerol (SAG), and 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG) possess similar potencies for PKC activation, but their effects are higher than 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG). Ziboh et al. (15) have also reported that in vivo stimulation of epidermal cells in the presence of -3 fatty acids can give rise to DAG-containing exogenous fatty acids that will modulate PKC activation and cell proliferation. In cardiomyocytes, addition of DHA has been shown to modulate cell contractility via the production of DAG containing this fatty acid and its subsequent action on PKC (16) . On the other hand, EPA incorporated into DAG has been found to reduce vascular complications associated with diabetes (17) . Though these studies have clearly demonstrated that DAGs that contain exogenous -3 fatty acids in place of AA modulate cell functions via PKC activation, no comparative study is available on the activation of different PKC isoenzymes by DAG containing -3/-6 fatty acids. It therefore seemed worthwhile to synthesize different molecular species of DAGs in which sn-2 position was substituted by either -6 or -3 fatty acids and to assess their effects on activation of different PKC isoforms in a cell-free system.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials
[³H]Arachidonic acid, [³H]docosahexaenoic acid, and [³H]eicosapentaenoic acid were purchased from NEN Products (Boston, MA). [-³²P]ATP (3000 Ci/mmol) was obtained from Isotopchim (Ganagobie, France). PKC substrate, the myelin basic protein (MBP), and recombinant protein kinase C isoforms (, ßI, , , and ) were obtained from Life Technology (Cergy Pontoise, France) and TEBU (Le Perray-en-Yvelines, France), respectively. The diacylglycerol (DiC8) was from Nu-Chek-Prep (Elysian; Minnesota, MN). Phosphatidyl-L-serine (PS) from bovine brain, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, and L--lysophosphatidylcholine, stearoyl were obtained from Sigma-Aldrich (St. Louis, MO). The composition of the PS was as follows: 0.8%, 20:4n-6; 1%, 20:5n-3; and 5.7%, 22:6n-3.

Synthesis of 1-stearoyl-2-arachidonoyl-, 1-stearoyl-2-docoshexaenoyl-, and 1-stearoyl-2-eicosapentaenoyl- sn-glycerol
1-Stearoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine(1-stearoyl-2-eicosapentaenoyl-PC) was synthesized as follows: eicosapentaenoic acid was esterified by L--lysophosphatidylcholine, stearoyl (lysoPC) by using rat liver microsomes in a reaction medium that contained the following 120 mM, Tris-HCl at pH 7.4; 3.2 mM, ATP; 1.56 mM, coenzyme A; 1.65 mM, MgCl₂; 156 mM, lysoPC; 62.5 mM, EPA and rat liver microsomes (0.8 mg/ml protein). The reaction was carried out at 37°C for 60 min and stopped by adding 2.5 volumes of methanol. 1-Stearoyl-2-eicosapentaenoyl-PC, was extracted using the method of Bligh and Dyer (18) .

1-Stearoyl-2-arachidonoyl-, 1-stearoyl-2-docosahexaenoyl-, and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol were obtained by the action of phospholipase C from Bacillus cereus on 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (1-stearoyl-2-arachidonoyl-PC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (stearoyl-2-docosahexaenoyl-PC), and 1-stearoyl-2-eicosapentaenoyl-PC, respectively, as described by Legrand et al. (19) . These DAGs were purified using straight-phase HPLC on a 30 cm µporasil column (Millipore, Saint-Quentin, France) and eluted isocratically with hexane/isopropyl alcohol, 100:1, v/v. The purified DAGs were identified by comparing their retention times with those of standard [³H]DAGs.

Standards of [³H]DAGs were obtained as follows: [³H]arachidonic acid, [³H]docosahexaenoic acid, and [³H]eicosapentaenoic acid were esterified by lysoPC using rat liver microsomes. The [³H]-labeled phospholipids obtained were hydrolyzed by phospholipase C and [³H]DAGs were purified using straight-phase HPLC as described above. HPLC-purified DAGs were quantified after transesterification at 80°C for 20 min by BF₃/methanol, using dinonadecanoin as internal standard. Fatty acid methyl esters were extracted with 2 ml hexane and separated by gas-liquid chromatography in a Packard Model 417 gas-liquid chromatograph equipped with a flame ionization detector and a 30-m capillary gas column coated with carbowax 20M. Analysis conditions were: oven at 194°C, injector and ionizing detector at 240°C. Helium was used as carrier gas, with a flow rate of 0.4 ml/min. Quantification of fatty acid peaks was achieved with reference to the internal standard by using DELSI ENICA 31 (Delsi Nermag; Rungis, France).

Preparation of vesicles
The required amounts of PS, DAG, and phorbol 12-myristate 13-acetate (PMA) in chloroform were dried under a stream of nitrogen in a glass tube and solubilized in Tris-HCl 20 mM by vortexing and sonication at 30°C for 5 min. Vesicles were stored at [m]80°C until using.

PKC assay
PKC activity was assayed by measuring the incorporation of ³²P from [-³²P]ATP into MBP as described by Vernhet et al. (20) . Individual isotypes of PKC were diluted with a buffer (20 mM, HEPES at pH 7.4; 100 mM, NaCl; 2 mM, EDTA; 2 mM, EGTA; 5 mM, DTT; 0.05%, Triton X-100, and 50%, glycerol). The reaction mixture (50 µl) contained 20 mM, Tris-HCl at pH 7.4; 20 mM, MgCl₂; 200 µg/ml, MBP; 20 mg/ml, PS; 50 ng, PKC. CaCl₂ at 1 µM (final concentration) was added in the reaction mixture for the Ca²⁺-dependent PKC activity assay. The reaction was initiated by the addition of 100 µM [-³²P]ATP (20–25 µCi/ml) for 10 min at 30°C and stopped by spotting 20 µl of samples on phosphocellulose filters that were later washed three times (10 min) with 1% H₃PO₄ and transferred to a scintillation counter to determine the radioactivity [³²P] MBP.

Statistical analysis
Results are shown as means ± SE for quadruplicate assay samples, reproduced independently at least twice. Statistical analysis of data was carried out using STATISTICA (version 4.1, Statsoft, Paris, France). The significance of the differences between mean values was determined by analysis of one-way ANOVA, followed by least significant differences test for individual comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Determination of experimental conditions for PKC activation
Sonic dispersion of lipids/vesicles and detergent-lipid mixed micellar preparations have both been used to determine the specificity and stoichiometry of PKC lipid–interaction (4) . In our study, we used the former method.

To determine the optimal concentration of PS to activate PKC, we used PS at increasing concentrations (Fig. 1 A). The fatty acid composition of the PS does not seem to be determinant in activation of PKC, as Bell and Sargent (21) found no significant differences in the activation of rat spleen protein kinase C whether the PS was from bovine brain (used in the present study) or trout liver, which lacks 20:4n-6 but contains 0.6% of 20:5n-3 and 43% of 22:6n-3.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of phosphatidylserine on activation of PKC. Lipid vesicles were prepared with increasing concentrations of phosphatidylserine in the absence or presence of dioctanoyl-glycerol (1.0 µM) as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with PS (5 µg/ml). A) *P < 0.001 or PS (5 µg/ml) + DiC8 (1.0 µM); B) *P < 0.001.

We observed that PS at 20 µg/ml exerts optimal stimulatory effects on PKC activation. Similarly, in the presence of DiC8, PS was found to stimulate optimal activation of PKC at the same concentration (Fig. 1B ). We therefore used PS at 20 µg/ml for further experiments.

In our study, we used DiC8 as a control in order to validate our experimental approach. Therefore, most of the comparisons have been made between three DAG species containing arachidonic acid (SAG), docosahexaenoic acid (SDG), or eicosapentaenoic acid (SEG). In the present study, DiC8 followed the pattern of SAG on PKC activation (Fig. 2 and Fig. 6 ).

View larger version (12K): [in this window] [in a new window]   Figure 2. Effects of different DAG species on activation of PKC. Lipid vesicles were prepared with different DAG molecular species (SAG, SEG, and SDG) and PKC activity was determined toward MBP in vesicles as a function of increasing concentrations of DAGs as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with SEG and SDG (*P<0.01).

View larger version (17K): [in this window] [in a new window]   Figure 6. Effects of different DAG species on activation of PKC. Lipid vesicles were prepared with different DAG molecular species (SAG, SEG, and SDG) and PKC activity was determined toward MBP in vesicles as a function of increasing concentrations of DAG as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with SEG and SDG (*P<0.001).

Effects of different DAG species on activation of cPKCs isoforms
Figure 2 shows that SAG exerted higher stimulatory effects on activation of PKC than SDG and SEG. Figure 3 shows that SDG and SEG are significantly better activators of PKCßI than SAG. However, there was not much significant difference in the activation of PKC by the three DAG species at all the concentrations (Fig. 4 ).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effects of different DAG species on activation of PKCßI. Lipid vesicles were prepared with different DAG molecular species (SAG, SEG, and SDG) and PKC activity was determined toward MBP in vesicles as a function of increasing concentrations of DAG as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with SEG and SDG (*P<0.01).

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of different DAG species on activation of PKC. Lipid vesicles were prepared with different DAG molecular species (SAG, SEG, and SDG) and PKC activity was determined toward MBP in vesicles as a function of increasing concentrations of DAG as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE.

Effects of different DAG species on activation of nPKCs isoforms
As to the activation of nPKC, we failed to establish a correlation for the activation of PKC isoform by different DAG species except for the concentration of 0.5 µM, where SAG was more efficient than SDG and SEG (Fig. 5 ). Furthermore, SAG-induced activation of PKC was significantly higher than with SDG and SEG (Fig. 6) .

View larger version (16K): [in this window] [in a new window]   Figure 5. Effects of different DAG species on activation of PKC. Lipid vesicles were prepared with different DAG molecular species (SAG, SEG, and SDG) and PKC activity was determined toward MBP in vesicles as a function of increasing concentrations of DAG as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with SEG and SDG (*P<0.001).

Competitive effects of DAGs containing -6 and -3 fatty acids on activation of PKC
We used the DAG molecular species at 0.5 µM, since at this concentration we observed the optimal effects of these agents on activation of most of the PKC isoforms (Figs. 2 3 4 5 6) .

Addition of SAG to vesicles containing either SEG or SDG exerts additive effects on activation of PKC without influencing PKCßI and (Fig. 7 B, C). SAG further significantly potentiated the activation of PKC, stimulated by SDG and SEG (Fig. 8 A). For PKC, however, SAG, diminished significantly SDG- and SEG-induced activation of this PKC isoform (Fig. 8B ).

View larger version (20K): [in this window] [in a new window]   Figure 7. Competition studies on DAG species and PMA on activation of cPKCs. Activation of PKC isoforms was essayed in the presence of CaCl2 (1.0 µM) with different DAG species (0.5 µM) in the presence or absence of PMA (1.6 µM) as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different vs. PMA (*P<0.001), SAG (£P<0.01), and SEG/SDG (P<0.001).

View larger version (20K): [in this window] [in a new window]   Figure 8. Competition studies on DAG species and PMA on activation of nPKCs. Activation of PKC isoforms was essayed in the absence of CaCl2 with different DAG species (0.5 µM) in the presence or absence of PMA (1.6 µM) as described in Materials and Methods. Data represent two independent experiments, each performed in quadruplicate and expressed as mean ± SE. Data are significantly different compared with PMA (*P<0.01), SAG (£P<0.01), and SEG/SDG (P<0.01).

Competitive effects of DAG species on PMA-induced activation of PKC
In these experiments, we used PMA at 1000 ng/ml because this concentration has been found to be optimal to activate PKC (22) . A perusal of Fig. 7 indicates that PMA stimulated higher activation of PKC than of PKCßI and PKC (PKC 1.5±0.02 vs. PKCßI 0.4±0.01 and PKC 0.14±0.005 nmol/min/mg protein, P<0.001). Figure 8 shows that PMA-induced activation was significantly higher in PKC than that in PKC (PKC 1.25±0.04 vs. PKC 0.27±0.003 nmol/min/mg protein, P<0.001).

On the other hand, SAG, SDG, and SEG diminished significantly the activation of PKCßI, , and , stimulated by PMA (Figs. 7 B, C and Fig. 8B ) but exerted no effect on PMA-induced activation of PKC and isoforms (Fig. 7A and Fig. 8A ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Accumulating evidence indicates that exogenous -3 PUFA supplied in the diet can give rise, ex vivo, to the production of DAGs from PIP₂ and PC containing these fatty acids (23 , 24) . In fact, earlier studies demonstrated that dietary -3 PUFA such as EPA and DHA are incorporated into PC, phosphatidylethanolamine (PE), PS, and phosphatidylinositol (PI) (25 , 26) . In platelets, however, it has been shown that after dietary fish oil supplementation, these two fatty acids are not incorporated into PI (27 , 28) ; this may be because platelets might selectively preserve the content of AA in PI for prostaglandin biosynthesis (29) .

The DAGs containing -3 fatty acids were found to modulate cell signaling differently from those containing -6 PUFA (9 , 30 , 31) . Albeit the DAG molecular species have been shown to modulate the activation of PKC (14) , no study has been conducted before on the modulation of different PKC isoforms by these agents. In the present study, we have synthesized three DAG molecular species in which sn-2 position contained either AA, DHA, or EPA and we have assessed their efficiency on activation of cPKCs (, ßI, ) and nPKCs (, ). We should recall that we did not include aPKC in our study, as enzymes of this subclass contain only one Cys-rich region within C1 domain and therefore cannot be activated by DAG and phorbol esters (1) .

In our study, we used the DiC8, a short and saturated fatty acid containing DAG, to validate our results on PKC activation. DiC8 is a cell-permeable analog of DAG and has been used to explore the cell signaling mechanisms involving the DAG/PKC pathway (32 , 33) . We observed that DiC8 followed the pattern of SAG to activate the different PKC isoforms. Our observations agree well with the findings of Goldberg et al. (34 , 35) , who have shown that DiC8 and SAG acts similarly on activation of PKC.

A perusal of activation of and ßI isoforms of cPKC subclass indicates that the degree of activation of the former was higher than that of the latter by all the molecular species of DAGs. Furthermore, SAG-induced activation of PKC was significantly higher than that induced by SEG and SDG. On the contrary, SAG-induced activation of PKCßI was significantly lower than that induced by the two DAGs containing -3 PUFAs (i.e., SEG and SDG). The differential activation of PKC and PKCßI by the three DAG species is not well understood as both of the enzymes belong to cPKCs. It is possible that PKC possesses higher affinity for SAG than SEG and SDG, and the opposite may be applicable for PKCßI isoforms. Since the experiments required the addition of Ca²⁺ for the activation of cPKCs, it is also possible that these DAG species may increase or decrease the apparent affinity of PKC for Ca²⁺ and PS, which may be responsible for the differential activation of the isotypes. Our hypothesis is partly substantiated by the observations of Goldberg et al. (35) , who have reported that the binding of different DAG species to PKC induces the conformational changes that modulate the binding of PS to its sites and subsequently influence the enzyme activation.

The three DAG species stimulated PKC to the same extent, and no significant difference was observed among SAG, SEG, and SDG. Though we did not use PUFAs in free form, our results agree closely with the observations by Shinomura et al. (36) , who have shown that -3 and -6 as free fatty acids stimulate activation of PKC to the same extent. The SAG-induced activation of PKC was higher than that induced by SEG and SDG only at 0.5 µM, whereas at all other concentrations no significant differences in the activation of this PKC isoform by the three DAG species were observed. The high stimulatory effects of SAG on PKC are specific, as DiC8 followed the same pattern. Activation of PKC by SAG was significantly higher than that by SEG and SDG at all the concentrations. Once again, the differences in the mechanism of action SAG, SEG, and SDG are not well understood.

We also conducted competition studies with the three DAG species and/or with PMA. We observed that addition of SAG to lipid vesicles containing SEG and SDG potentiated the activation of PKC and PKC, but not of ßI and , isoforms compared with vesicles containing SEG and SDG alone. On the contrary, the activation of PKC was significantly diminished by the addition of SAG to vesicles containing SEG and SDG. How the different DAG molecular species compete with each other either positively or negatively remains unexplained. However, it is possible that there are different binding sites for different DAG molecular species, as suggested by Alpert et al. (37) . These investigators observed that in vitro cell-generated DAG-HETE inhibited the action of endogenous DAG on activation of PKC in human tracheal epithelial cells; they also proposed that PKC isoforms possess different binding sites, destined to two molecular species of DAG (37) .

The addition of the three DAG molecular species to vesicles containing PMA either exerted no effect on activation of PKC and PKC or diminished significantly the activation of PKCßI, PKC, and PKC. It is possible that DAG and PMA are exerting their effects via two different binding sites present on PKC. Our hypothesis can be supported by the observations of Slater et al. (38 , 39) , who have demonstrated that cPKC and nPKC bear two distinct sites for PMA. These investigators have further shown there are low- and high-affinity binding sites for PMA within C1 domain of the enzyme, and DAG may either inhibit or enhance the binding of PMA to, respectively, low- and high-affinity binding sites (39) . It is possible that the three DAG molecular species in our study competed with PMA at low-affinity binding sites present on ßI, , and isoforms of PKC, resulting in the inhibition of enzyme activation induced by phorbol esters.

Our findings show, for the first time, that the different isoforms of cPKCs and nPKCs differ in their responses to DAGs containing AA, EPA, or DHA. We speculate that similar modulation of PKC activation may occur in vivo, after receptor activation during an exogenous supply of these fatty acids, and this phenomenon may be implicated in progression of different pathologies. Further studies of the in vivo production of these DAG molecular species and their action on activation of different isoforms of PKC in health and disease are awaited.

	ACKNOWLEDGMENTS

 
This work was supported by a contingent grant from Burgundy Region, Dijon and Group Lipids and Nutrition, Paris (France).

Received for publication June 12, 2001. Revision received August 21, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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