Total Diacylglycerol Levels Research Articles

Functional relationship between sphingomyelin synthase‐related protein and diacylglycerol kinase δ

IntroductionThe δ isozyme of diacyglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). Intriguingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐ and/or palmitoleic acid (16:1)‐containing DG molecular species in response to high glucose stimulation in myoblasts. However, it is still unclear where 16:0‐ and/or 16:1‐containing DG molecular species come from, although another species arachidonic acid (20:4)‐containing DGs are derived from phosphatidylinositol (PI) turnover. Sphingomyelin synthase (SMS) 1 and SMS‐related protein (SMSr) synthesize the sphingomyelin and ceramide‐phosohoethanolamine by the transfer of phosphocholine/phosphoethanolamine to ceramide, respectively. These are also assumed to function as DG‐generating enzymes. SMS1, SMSr and DGKδ contain a sterile α motif domain (SAM), which is a putative protein interaction module. Interestingly, we found that DGKδ interacted with SMSr via their SAMs directly. In the present study, we investigated the functional relationship between DGKδ and SMSr.Results and ConclusionWe first investigated the changes in the amounts of PA molecular species in COS‐7 cells overexpressing DGKδ and/or SMSr using LC‐MS/MS. We found that the overexpression of SMSr and DGKδ significantly enhanced the production of 16:0‐ and/or 16:1‐containing PA species such as 16:0/16:1‐, 16:0/16:0‐, 16:0/18:1‐ and 16:0/18:2‐PA in COS‐7 cells. It is possible that DGKδ and SMSr are functionally linked and that SMSr acts in an upstream pathway of DGKδ by providing 16:0‐ and/or 16:1‐DG. To support the possibility, we determined DG levels in SMSr‐overexpressing cells. Compared with control cells, total DG levels in SMSr‐overexpressing cells were significantly increased. In particular, 16:0‐ and/or 16:1‐containig DG species, such as 14:0/16:0 (30:0)‐, 16:0/16:1 (32:1)‐, 16:1/18:2 (34:3)‐, and 16:1/18:1 (34:2)‐DG were significantly increased. These results further suggest the functional relationship (SMSr supplies DG to DGKδ) between DGKδ and SMSr. To examine whether forming SMSr‐DGKδ heteromeric complex affects DGKδ activity, we measured purified DGKδ activity in vitro using DG and ATP as substrates. Interestingly, when presence of purified SMSr with purified DGKδ, DGK activity was significantly increased. However, the deletion of SAM of SMSr did not significantly augment the DGKδ activity. Taken together, these results suggest that SMSr is one of the candidates of upstream DG‐providing enzymes of DGKδ and that SMSr enhances DGKδ activity by forming heteromeric complex via their SAMs.Support or Funding InformationThis work was supported in part by grants from MEXT/JSPS KAKENHI (Grant Numbers: JP18J20003 to C.M.); The Chiba Bank (to C.M.); the Chiba University Venture Business Laboratory (to C.M.); Chiba University Open Recruitment for International Exchange Program at the Institute for Global Prominent Research (to C.M.); and the American Society for Biochemistry and Molecular Biology (ASBMB 2019 Graduate/Postdoctoral Travel Award to C.M)

ERK3 Interacts with DGKζ and Alters Signaling Lipids in Lung Cancer Cells

Extracellular‐Regulated Kinase 3 (ERK3) promotes migration and invasion in many cancers, including lung cancer. However, the mechanisms by which this atypical Mitogen‐Activated Protein Kinase (MAPK) induces these effects remain poorly understood. The p21‐activated kinase (PAK)‐Rac1 complex, which can be activated by phosphatidic acid, is associated with cell migration and has been shown to activate ERK3. Here, we show ERK3 also binds to phosphatidic acid and treatment of cells with phosphatidic acid results in an increase in ERK3 phosphorylation. Intriguingly, we have also found ERK3 modulates Rac1 activity and PAK phosphorylation, suggesting a positive feedback loop. In ERK3 knockdown cells, total diacylglycerol level was elevated while total phosphatidic acid level was decreased. In a yeast two‐hybrid assay, ERK3 was shown to interact with Diacylglycerol Kinase ζ (DGKζ), an enzyme which converts diacylglycerol to phosphatidic acid. This interaction has been confirmed by co‐immunoprecipitation and appears to occur through the C34 domain of ERK3. By immunofluorescence, these proteins were shown to co‐localize in cells with ERK3 enhancing cytoplasmic localization of DGKζ. Together, these data suggest ERK3 may regulate DGKζ to facilitate phosphatidic acid production, thereby activating the Rac1‐PAK complex and inducing cell migration.Support or Funding InformationNCI 1R01 CA193264‐01Wright State University start‐up fund

Endurance exercise training and high-fat diet differentially affect composition of diacylglycerol molecular species in rat skeletal muscle.

Insulin resistance of peripheral muscle is implicated in the etiology of metabolic syndrome in obesity. Although accumulation of glycerolipids, such as triacylglycerol and diacylglycerol (DAG), in muscle contributes to insulin resistance in obese individuals, endurance-trained athletes also have higher glycerolipid levels but normal insulin sensitivity. We hypothesized that the difference in insulin sensitivity of skeletal muscle between athletes and obese individuals stems from changes in fatty acid composition of accumulated lipids. Here, we evaluated the effects of intense endurance exercise and high-fat diet (HFD) on the accumulation and composition of lipid molecular species in rat skeletal muscle using a lipidomic approach. Sprague-Dawley female rats were randomly assigned to three groups and received either normal diet (ND) in sedentary conditions, ND plus endurance exercise training, or HFD in sedentary conditions. Rats were fed ND or HFD between 4 and 12 wk of age. Rats in the exercise group ran on a treadmill for 120 min/day, 5 days/wk, for 8 wk. Soleus muscle lipidomic profiles were obtained using liquid chromatography/tandem mass spectrometry. Total DAG levels, particularly those of palmitoleate-containing species, were increased in muscle by exercise training. However, whereas the total DAG level in the muscle was also increased by HFD, the levels of DAG molecular species containing palmitoleate were decreased by HFD. The concentration of phosphatidylethanolamine molecular species containing palmitoleate was increased by exercise but decreased by HFD. Our results indicate that although DAG accumulation was similar levels in trained and sedentary obese rats, specific changes in molecular species containing palmitoleate were opposite.

The impact of obesity in the cardiac lipidome and its consequences in the cardiac damage observed in obese rats

AimsTo explore the impact of obesity on the cardiac lipid profile in rats with diet-induced obesity, as well as to evaluate whether or not the specific changes in lipid species are associated with cardiac fibrosis. MethodsMale Wistar rats were fed either a high-fat diet (HFD, 35% fat) or standard diet (3.5% fat) for 6 weeks. Cardiac lipids were analyzed using by liquid chromatography-tandem mass spectrometry. ResultsHFD rats showed cardiac fibrosis and enhanced levels of cardiac superoxide anion (O2), HOMA index, adiposity, and plasma leptin, as well as a reduction in those of cardiac glucose transporter (GLUT 4), compared with control animals. Cardiac lipid profile analysis showed a significant increase in triglycerides, especially those enriched with palmitic, stearic, and arachidonic acid. An increase in levels of diacylglycerol (DAG) was also observed. No changes in cardiac levels of diacyl phosphatidylcholine, or even a reduction in total levels of diacyl phosphatidylethanolamine, diacyl phosphatidylinositol, and sphingomyelins (SM) was observed in HFD, as compared with control animals. After adjustment for other variables (oxidative stress, HOMA, cardiac hypertrophy), total levels of DAG were independent predictors of cardiac fibrosis while the levels of total SM were independent predictors of the cardiac levels of GLUT 4. ConclusionsThese data suggest that obesity has a significant impact on cardiac lipid composition, although it does not modulate the different species in a similar manner. Nonetheless, these changes are likely to participate in the cardiac damage in the context of obesity, since total DAG levels can facilitate the development of cardiac fibrosis, and SM levels predict GLUT4 levels.

The impact of obesity in the cardiac lipidome and its consequences in the cardiac damage observed in obese rats

AimsTo explore the impact of obesity on the cardiac lipid profile in rats with diet-induced obesity, as well as to evaluate whether or not the specific changes in lipid species are associated with cardiac fibrosis. MethodsMale Wistar rats were fed either a high-fat diet (HFD, 35% fat) or standard diet (3.5% fat) for 6 weeks. Cardiac lipids were analyzed using by liquid chromatography-tandem mass spectrometry. ResultsHFD rats showed cardiac fibrosis and enhanced levels of cardiac superoxide anion (O2), HOMA index, adiposity, and plasma leptin, as well as a reduction in those of cardiac glucose transporter (GLUT 4), compared with control animals. Cardiac lipid profile analysis showed a significant increase in triglycerides, especially those enriched with palmitic, stearic, and arachidonic acid. An increase in levels of diacylglycerol (DAG) was also observed. No changes in cardiac levels of diacyl phosphatidylcholine, or even a reduction in total levels of diacyl phosphatidylethanolamine, diacyl phosphatidylinositol, and sphingomyelins (SM) was observed in HFD, as compared with control animals. After adjustment for other variables (oxidative stress, HOMA, cardiac hypertrophy), total levels of DAG were independent predictors of cardiac fibrosis while the levels of total SM were independent predictors of the cardiac levels of GLUT 4. ConclusionsThese data suggest that obesity has a significant impact on cardiac lipid composition, although it does not modulate the different species in a similar manner. Nonetheless, these changes are likely to participate in the cardiac damage in the context of obesity, since total DAG levels can facilitate the development of cardiac fibrosis, and SM levels predict GLUT4 levels.

Signaling at the membrane interface by the DGK/SK enzyme family

The sphingosine (SK) and diacylglycerol (DGK) kinases have become the subject of considerable focus recently due to their involvement as signaling enzymes in a variety of important biological processes. These lipid signaling kinases are closely related by sequence as well as functional properties. These enzymes are soluble, yet their substrates are hydrophobic. Therefore, they must act at the membrane interface. Second, for both of these enzyme families, their substrates (diacylglycerol for DGKs, sphingosine for SKs) as well as their products (phosphatidic acid for DGK, sphingosine-1-phosphate for SK) have signaling function. To understand how the signaling processes emanating from these kinases are regulated it is critical to understand the fundamental mechanisms that control their enzymatic activity. This is particularly true for the rational design of small molecules that would be useful as therapeutic compounds. Here we summarize enzymological properties of the diacylglycerol and SKs. Further, because the three-dimensional structure of the eukaryotic members of this family has yet to be determined, we discuss what can be gleaned from the recently reported structures of related prokaryotic members of this enzyme family.

D-α-Tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity

We have reported that d-α-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from α-tocopherol’s principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of α-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of α-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of α-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-α-tocopherol’s inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40±10% (mean±S.E.) ( P<0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-α-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-α-tocopherol was also induced in a time- and dose-dependent manner. DAG kinase activity was increased by 57±19% ( P<0.05) in human VSMC and 112±35% ( P<0.05) in rat VSMC after 24 h of incubation with d-α-tocopherol (100 μg/ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124±34%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-α-tocopherol treatment. These results provide strong and detailed evidence that d-α-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect.

Effect of Eicosapentaenoic Acid on Glucose-Induced Diacylglycerol Synthesis in Cultured Bovine Aortic Endothelial Cells

Hyperglycemia has been postulated to increase diacylglycerol (DAG) level through de novo synthesis pathway and subsequently activate protein kinase C (PKC) in vascular cells, possibly leading to vascular dysfunction associated with diabetes. In this study, we examined the effect of eicosapentaenoic acid (EPA) on high glucose-induced increase in DAG level in cultured aortic endothelial cells (ECs). In ECs, total DAG level was significantly increased in the cells cultured with high glucose levels (400 mg/dl) compared with the cells with normal glucose levels (100 mg/dl). The addition of EPA completely prevented high glucose-induced increase in total DAG level. In contrast, other common fatty acids such as palmitate and oleate significantly stimulated DAG syntheisis, although arachidonate did not affect it. High glucose level significantly stimulated the incorporation of3H-palmitate into DAG, while it did not affect the incorporation of3H-arachidonate into DAG. The addition of EPA completely prevented the high glucose-induced increase in3H-palmitate incorporation into DAG, while it did not affect the3H-arachidonate incorporation. These findings suggest that EPA can prevent high glucose induced-increase in DAG level in ECs, probably by specifically inhibiting de novo synthesis at the step of acylation. EPA may be one of the candidates for clinical agents normalizing activation of DAG-PKC pathway in diabetic vascular tissues and preventing vascular complications associated with diabetes.

Prevention of diabetes-induced abnormal retinal blood flow by treatment with d-alpha-tocopherol.

Hyperglycemia in diabetes mellitus has been shown to activate diacylglycerol (DAG)-protein kinase C (PKC) pathway in the vascular tissues, possibly altering vascular function. We have characterized the effects of vitamin E (d-alpha-tocopherol) on activation of PKC and DAG levels in retinal tissues of diabetic rats, and correlated its effects to retinal hemodynamics using video-based fluorescein angiography (VFA). Comparing streptozotocin-induced diabetic rats to controls, membranous PKC specific activities were increased by 71% (p < 0.05). Western blot analysis showed that the membranous PKC beta II isoform was significantly increased by 133 +/- 45% (p < 0.05). Intraperitoneal injection of d-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membranous PKC specific activity and PKC beta II protein shown by immunoblots. Similar to PKC activities, total DAG levels were increased in the retina and were normalized by d-alpha-tocopherol treatment. Physiologically, abnormalities of retinal blood hemodynamics, as measured using VFA, which previously have been reported to be associated with increases of DAG and PKC levels in the diabetic rats, were prevented by d-alpha-tocopherol treatment in diabetic rats. The direct effect of d-alpha-tocopherol on total DAG and [3H]-palmitate incorporation into DAG were also examined using cultured bovine retinal endothelial cells (REC). Exposure of REC to 22 mM glucose for three days increased total DAG and [3H]-palmitate labeled DAG levels by 35 +/- 8% and 50 +/- 8%, respectively (p < 0.05). The presence of d-alpha-tocopherol (50 micrograms/ml) prevented the increase of both total DAG and [3H]-palmitate labeled DAG levels in cells exposed to 22 mM glucose. These findings suggested that the mechanism of the d-alpha-tocopherol's effect appears to be mediated by the normalization of the hyperglycemia-induced activation of the DAG-PKC pathway which leads to the normalization of abnormal retinal blood flow seen in diabetes mellitus.

Substitution of 15(S)hydroxyeicosatetraenoic acid in phosphatidylinositol alters the growth of liver epithelial cells

We investigated the substitution of 15(S)-hydroxyeicosatetraenoic acid (15(S)HETE) in phospholipid signalling pathways and its consequences on the growth of non-transformed (NT-) and spontaneously transformed (T-) rat liver epithelial cells (RLEC). 15(S)HETE was selectively incorporated into the sn-2 position of phosphatidylinositol (PI) and at a higher rate into T-RLEC. RLEC rapidly mobilized the resulting 15(S)HETE-containing PI (15(S)HETE-PI) and produced 1-acyl,2-[15(S)HETE]-glycerol. Although total diacylglycerol levels were similar in both cell types, the ratio 1-acyl,2-[15(S)HETE]-glycerol/15(S)HETE-PI was higher in NT-RLEC, suggesting a lower mobilization of 15(S)HETE-PI in T-RLEC. Using rat brain protein kinase C, 1-stearoyl,2-[15(S)HETE]-glycerol was as potent an in vitro protein kinase C activator as 1-stearoyl,2-arachidonoyl-glycerol. Finally, selective substitution of 15(S)HETE in PI altered DNA synthesis in T-RLEC: whereas low concentrations of 15(S)HETE (1 nM and 10 nM) in these cells were mitogenic, higher concentrations resulted in a 30% inhibition of DNA synthesis.

Preliminary characterization of diacylglycerol generation in human basophils: temporal relationship to histamine release and resolution of degranulation.

Purified human basophils were examined for changes in diacylglycerol levels to determine whether the transient nature of a N-formyl-methionyl-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protein kinase C (PKC) activity could be explained by the transient production of diacylglycerol (DAG). In preliminary experiments total DAG levels were measured by the DAG kinase assay. Although elevations followed stimulation with 1 microM fMLP (basal levels of 15 pmol/10(6) basophils vs. 45 pmol/10(6) basophils at the 3-min time point), there were no detectable changes in the first 60 s of the reaction. Histamine release is typically complete by 30-45 s. Measurement of inositol trisphosphate indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils. If DAG were produced at similar levels, the DAG kinase assay would not have detected the elevation. Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA-DAG) and 1-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negative ion chemical ionization mass spectroscopy). A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inositol trisphosphate levels and occurring during the period of histamine release. However, a much more pronounced second phase to the SA-DAG response also occurred, mirroring the total DAG levels. This second phase of the DAG response, either total or SA-DAG, was transient on a time scale temporally coincident with the appearance and resolution of degranulation sacs as measured by fluorescence microscopy. These data suggest that there is selective generation of DAG species in the early reaction and the later appearance of DAG may be related to the formation and resolution of granule structures that follow the secretion of histamine.

Vitamin E normalizes diacylglycerol-protein kinase C activation induced by hyperglycemia in rat vascular tissues.

Hyperglycemia, a major cause of vascular complications in diabetes, has been shown to activate the diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular tissues. We have found that D-alpha-tocopherol (vitamin E) treatment reversed the adverse effects of hyperglycemia both in vitro and in vivo. In aortic smooth muscle cells (ASMCs), the PKC specific activity from the membranous fraction and total DAG were increased by 31 +/- 4% (P < 0.05) and 50 +/- 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/l. D-alpha-tocopherol prevented the glucose-stimulated increases in DAG level and PKC activity as well as the amount of PKC beta II isoform in ASMCs cultured with elevated glucose levels. Comparing streptozotocin-induced diabetic rats after 2 weeks of disease to controls, specific membranous PKC activities and total DAG levels were increased in aorta (162%, P < 0.05; 60%, P < 0.05). Intraperitoneal injection of D-alpha-tocopherol (40 mg/kg) every other day resulted in a significant decrease of the elevated membranous PKC specific activity and total DAG levels in parallel with a significant increase of D-alpha-tocopherol content in the aorta. These findings suggested that D-alpha-tocopherol can prevent the activation of PKC in the vascular cells and aorta induced by hyperglycemia by normalizing the elevated levels of DAG.

Analysis of R59022 actions in Xenopus laevis oocytes.

R59022, a diacylglycerol (DAG) kinase inhibitor, stimulated meiotic maturation of Xenopus laevis oocytes when applied extracellularly. The time course of R59022-induced oocyte maturation was proportional to the concentration of R59022 in the low micromolor range, and the 30 microM-induced response was a fast or faster than progesterone-induced maturation. Dose-response analysis yielded an apparent EC50 for R59022-induced oocyte maturation of approximately 15 microM. An increase in total oocyte DAG levels was observed following treatment with 10 microM R59022. Treatment of oocytes with R59022 also resulted in a significant increase in intracellular pH similar to the increase observed with progesterone. When various phosphodiesterase (PDE) inhibitors were tested for their effects on R59022-induced oocyte maturation, papaverine (a potent nonselective inhibitor of PDE) and CI-930 (a selective PDE III inhibitor) were observed to significantly inhibit the R59022-stimulated response. The sensitivity of R59022-induced oocyte maturation to inhibition by papaverine was intermediate between the sensitivities of the IGF-1- or progesterone-induced responses. Treatment of oocytes with R59022 did not significantly affect the level of oocyte PDE activity measured in vivo, suggesting that elevated levels of DAG may parallel observed increases in PDE but do not directly lead to a stimulation of PDE. The t ime course for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation by progesterone rather than IGF-1. Treatment of isolated membranes with R59022 resulted in inhibition of membrane-associated adenyl cyclas e activity that was not mimicked by DAG analogs. Thus, in addition to elevating oocyte levels of DAG, R59022 also has steroid-like actions.

Normalization of Diacylglycerol-Protein Kinase C Activation by Vitamin E in Aorta of Diabetic Rats and Cultured Rat Smooth Muscle Cells Exposed to Elevated Glucose Levels

Hyperglycemia and diabetes have been shown to increase diacylglycerol (DAG) level and activate protein kinase C (PKC) activity in the vascular tissues, possibly altering vascular function. We have characterized the effects of D-alpha-tocopherol (vitamin E) on PKC activities and DAG levels in rat aortic smooth muscle cells (ASMCs) cultured with elevated glucose levels as well as in the vascular tissues obtained from control and diabetic rats. In ASMCs, the specific PKC activity from the membraneous fraction and total DAG level were increased by 31 +/- 4% (P < 0.05) and 50 +/- 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/l. The addition of D-alpha-tocopherol and another lipophilic antioxidant, probucol, prevented the glucose-stimulated increases in DAG level and PKC activity. By immunoblotting studies, D-alpha-tocopherol treatment was able to reduce the enhancement of PKC beta II isoform in the membraneous fraction isolated from ASMCs. Comparing streptozotocin-induced diabetic rats with their nondiabetic controls, both membraneous-specific PKC activities and total cellular DAG levels were increased in aorta by 162% (P < 0.05) and 60% (P < 0.05), respectively. Intraperitoneal injection of D-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membraneous-specific PKC activities and total DAG levels in parallel with a significant increase of D-alpha-tocopherol contents in the aorta and plasma. These findings have demonstrated that D-alpha-tocopherol can prevent the activation of PKC activities in the vascular cells and tissues induced by hyperglycemia by lowering DAG levels, possibly via its antioxidant effect.

Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation

The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats. With diabetes, all the isoenzymes and the total DAG level were increased in the rat retina, whereas no changes were found in the rat brain. Insulin treatment normalized plasma glucose levels and partially prevented the increases in the membraneous PKC activity and all the isoenzymes in the retina. In the retinal endothelial cells, the total DAG level and PKC specific activities are increased by 36 and 22%, respectively, in the membraneous pool when the glucose levels are changed from 5.5 to 22 mM. Activation of PKC activity and isoform beta II by the vitreal injection of phorbol dibutyrate mimicked the abnormal retinal blood circulation observed in diabetic rats (2.22 +/- 0.24 vs. 1.83 +/- 0.40 s). Thus diabetes and elevated glucose levels will increase DAG level and PKC activities and its isoenzyme specifically in vascular cells and may affect retinal hemodynamics.

Effect of harvesting methods, growth conditions and growth phase on diacylglycerol levels in cultured human adherent cells

The cellular mass of sn-1,2-diacylglycerols, which are intracellular second messengers which activate protein kinase C, were quantitatively determined with an enzymatic assay. The method employed to harvest cultured human skin fibroblasts or human epidermal A431 cells prior to extraction of lipid into chloroform/methanol affected diacylglycerol (DAG) levels. Scraping or trypsinization significantly increased DAG levels. A method was devised to allow reliable and reproducible DAG measurements from adherent cells. The addition of methanol prior to scraping was shown to stop cellular metabolism and to permit accurate quantitation. Importantly, this solvent was compatible with cultures grown on plastic. Using this method, growth conditions which could affect DAG levels were investigated. Changes in the osmolality of the culture medium did not affect the DAG levels of A431 cells; exposure of A431 cells to acidic pH or elevated temperature lowered DAG levels. In contrast to fibroblasts, the total DAG levels of A431 cells continued to increase during serum deprivation. The highest DAG levels, normalized to phospholipids, were observed during the exponential growth phase. This ratio dropped when the cultures reached confluency. These experiments also demonstrated that A431 cells possess higher DAG levels than do normal fibroblasts. The function of DAG in cellular regulation is discussed.